阅读说明：本技术 针对抗病性状筛选植物原生质体 (Screening plant protoplast for disease resistance ) 是由 T·A·莱恩伯格 V·L·S·库尔兹 于 2019-07-12 设计创作，主要内容包括：提供了用于针对抗病性状筛选植物细胞(特别是植物原生质体)的方法,以及用于执行此类方法的试剂盒。该方法在微流体装置中进行,该微流体装置包括适于培养和筛选植物原生质体的至少一个生长室和流动区域。微流体芯片的生长室的至少一个表面可以包括共价连接的涂覆材料或表面改性配体。该试剂盒可以包括微流体芯片以及用于检测植物原生质体的活力的试剂(可选地,表面调理剂或表面改性剂)。(Methods for screening plant cells, particularly plant protoplasts, for disease resistance traits and kits for performing such methods are provided. The method is performed in a microfluidic device comprising at least one growth chamber and a flow region suitable for culturing and screening plant protoplasts. At least one surface of the growth chamber of the microfluidic chip may include a covalently attached coating material or surface modifying ligand. The kit may include a microfluidic chip and reagents (optionally, surface conditioners or surface modifiers) for detecting viability of plant protoplasts.)

1. A method of identifying plant protoplasts that lack pathogen resistance, the method comprising:

introducing a first fluid medium comprising one or more protoplasts into a microfluidic device comprising a housing having a flow region and at least one growth chamber;

Moving a first protoplast of the one or more protoplasts into a first growth chamber of the at least one growth chamber;

contacting the first protoplast with a pathogen; and

monitoring viability of the first protoplast during a first period of time after contacting the first protoplast with the pathogen,

wherein the viability of the protoplasts at the end of the first period of time indicates that the protoplasts lack resistance to the pathogen.

2. The method of claim 1, wherein the one or more protoplasts are from a large area crop.

3. The method of claim 2, wherein the large area crop is a wheat, corn, soybean, or cotton plant.

4. The method of claim 1, wherein the one or more protoplasts are from a high value crop or an ornamental crop.

5. The method of claim 4, wherein the high value crop is a tomato, lettuce, pepper or squash plant.

6. The method of claim 1, wherein the one or more protoplasts are from a turf or a forage plant.

7. The method of claim 6, wherein the turf or forage plant is a grass or alfalfa plant.

8. The method of claim 1, wherein the one or more protoplasts are from a test plant.

9. The method of any one of claims 1 to 8, wherein the pathogen is a plant pathogen or a molecule derived from a plant pathogen.

10. The method of claim 9, wherein the plant pathogen is a virus, a bacterium, or a fungal cell.

11. The method of claim 9, wherein the pathogen is a molecular agent or a fragment of a molecular agent.

12. The method of any one of claims 1 to 8, wherein contacting the first protoplast with the pathogen comprises flowing a second fluid medium comprising the pathogen into the flow region of the microfluidic device.

13. The method of claim 12, wherein contacting the first protoplast with the pathogen further comprises moving the pathogen into the isolated region of the first growth chamber or allowing the pathogen to diffuse from the flow region to the isolated region of the first growth chamber.

14. The method of any one of claims 1 to 8, wherein the housing further comprises a base, a microfluidic circuit structure disposed on the base, and a lid.

15. The method of claim 14, wherein the lid and the base are part of a dielectrophoretic DEP mechanism for selectively inducing DEP forces on small objects, and wherein moving the first protoplast into the first growth chamber comprises applying DEP forces on the first protoplast.

16. The method of any one of claims 1 to 8, wherein the microfluidic device further comprises a first electrode, an electrode-activated substrate, and a second electrode, wherein the first electrode is part of a first wall of the housing, and the electrode-activated substrate and the second electrode are part of a second wall of the housing, wherein the electrode-activated substrate comprises a photoconductive material, a semiconductor integrated circuit, or a phototransistor, and wherein moving the first protoplast into the first growth chamber comprises applying a DEP force on the first protoplast.

17. The method of claim 16, wherein the first wall is a lid, and wherein the second wall is a base.

18. The method of claim 16, wherein the electrode activation substrate comprises a phototransistor.

19. The method of claim 16, wherein the cover and/or the base are optically transparent.

20. The method of any one of claims 1 to 8, wherein the first growth chamber is an isolation pen comprising an isolation region and a communication region that fluidly communicates the isolation region to the flow region, and wherein the isolation region is an unswept region of the microfluidic device.

21. The method of claim 20, wherein the housing further comprises a microfluidic channel comprising at least a portion of the flow region, wherein the communication region of the isolation fence comprises a proximal opening to the microfluidic channel and a distal opening to the isolation region, the proximal opening having a width W_conIs about 50 microns to about 150 microns, and wherein the length L of the communication zone from the proximal opening to the distal opening_conIs the width W of the proximal opening of the communication region_conAt least 1.0 times.

22. The method of claim 21, wherein the length L of the communication region from the proximal opening to the distal opening _conIs the width W of the proximal opening of the communication region_conAt least 1.5 times.

23. The method of claim 21, wherein the length L of the communication region from the proximal opening to the distal opening_conIs the width W of the proximal opening of the communication region_conAt least 2.0 times.

24. The method of claim 21, wherein the proximal opening of the communication region has a width W_conFrom about 50 microns to about 100 microns.

25. The method of claim 21, wherein the length L of the communication region from the proximal opening to the distal opening_conBetween about 50 microns and about 500 microns.

26. The method of claim 21, wherein the height H of the microfluidic channel at the proximal opening of the communication region_chBetween 20 microns and 100 microns.

27. The method of claim 21, wherein the microfluidic channel has a width W at the proximal opening of the communication region_chBetween about 50 microns and about 500 microns.

28. The method of claim 20, wherein the isolated region of the isolation fence has a volume of about 5 x 10⁵To about 5X 10⁶Cubic microns.

29. The method of claim 20, wherein the isolated region of the isolation fence has a volume of about 1 x 10 ⁶To about 2X 10⁶Cubic microns.

30. The method of claim 20, wherein the proximal opening of the communication region is parallel to a direction of bulk flow in the flow region.

31. The method of any one of claims 1 to 8, wherein monitoring viability of the first protoplast during the first time period comprises monitoring cell division of the first protoplast, and wherein cell division of the first protoplast indicates a lack of resistance of the protoplast to the pathogen.

32. The method of any one of claims 1 to 8, wherein monitoring viability of the first protoplast during the first time period comprises: maintaining the microfluidic chip at a temperature of about 20 ℃ to about 30 ℃ during the first period of time, and/or minimizing exposure of the first protoplast during the first period of time.

33. The method of any one of claims 1 to 8, wherein monitoring viability of the first protoplast during the first time period comprises: periodically perfusing a protoplast growth medium through a flow region of the microfluidic device during the first period of time.

34. The method of claim 33, wherein the protoplast growth medium is perfused through the flow region no more than once every three days.

35. The method of any one of claims 1 to 8, wherein monitoring viability of the first protoplast during the first time period comprises: staining the first protoplast with a cell viability dye.

36. The method of any one of claims 1 to 8, wherein monitoring viability of the first protoplast during the first time period comprises: staining the first protoplast by a chlorophyll stain and/or a cell wall stain.

37. The method of any one of claims 1 to 8, wherein the first period of time is at least 12 hours.

38. The method of claim 37, wherein the first period of time is at least 96 hours.

39. The method of any of claims 1 to 8, further comprising:

determining that the first protoplast lacks resistance to the pathogen; and

outputting the first protoplast from the first growth chamber and the microfluidic device.

40. The method of any of claims 1 to 8, further comprising:

Determining that the first protoplast lacks resistance to the pathogen; and

sequencing one or more disease resistance genes of the first protoplast.

41. The method of any of claims 1 to 8, further comprising:

determining that the first protoplast lacks resistance to the pathogen; and

sequencing the transcriptome of the first protoplast.

42. The method of any of claims 1 to 8, further comprising:

determining that the first protoplast lacks resistance to the pathogen; and

sequencing the genome of the first protoplast.

43. The method of claim 40, further comprising:

identifying a molecular change or defect in the sequence: sequences of one or more disease resistance genes, transcriptomes, and/or genomes associated with a lack of pathogen resistance.

44. The method of any of claims 1-8, further comprising:

moving at least one protoplast into each of a plurality of growth chambers in the microfluidic device; and

performing the remaining steps of the method for each protoplast that is moved into the plurality of growth chambers.

45. A kit for screening plant protoplasts for a disease-resistant trait, the kit comprising:

A microfluidic chip, wherein the microfluidic chip comprises a housing having a flow region and at least one growth chamber; and

a reagent for detecting the viability of the plant protoplasts.

46. The kit of claim 45, further comprising a surface conditioning agent.

47. The kit of claim 45, further comprising a conditioning modifier, and wherein at least one surface of the growth chamber comprises a surface modifying ligand.

48. The kit of claim 45, wherein at least one surface of the growth chamber comprises a covalently attached coating material.

49. The kit of any one of claims 45 to 48, wherein the reagent for detecting viability of the plant protoplasts is a fluorescent stain.

Background

In the field of bioscience and related fields, it may be useful to culture cells (particularly single cells) under conditions that allow for monitoring and/or determination of the cells, so that the cells of interest may be isolated for further research or use. Unfortunately, for most types of cells, suitable culture conditions are still unknown or not optimized. Some embodiments disclosed herein include a process of culturing plant protoplasts in a microfluidic device. Protoplasts can be cultured individually or in groups. Other embodiments disclosed herein include a process of screening plant protoplasts for a desired trait (e.g., a disease resistance gene) while culturing the protoplasts in a microfluidic device.

Disclosure of Invention

In one aspect, a method of identifying plant protoplasts that lack pathogen resistance is disclosed. The method can comprise the following steps: introducing a first fluid medium comprising one or more plant protoplasts into a microfluidic device comprising a housing having a flow region and at least one growth chamber; moving a first protoplast of the one or more protoplasts into a first growth chamber of the at least one growth chamber; contacting the first protoplast with a pathogen; and monitoring the viability of the first protoplast during a first time period after contacting the first protoplast with the pathogen. The viability of the protoplasts at the end of the first period of time indicates that the protoplasts lack resistance to the pathogen. Such protoplasts can be exported from their corresponding growth chambers and recovered off-chip for further analysis (e.g., sequencing to determine the molecular basis for lack of pathogen resistance). In certain embodiments, the method further comprises moving at least one protoplast into each of a plurality of growth chambers in the microfluidic device and performing the remaining steps of the method for all protoplasts moved into the plurality of growth chambers.

In certain embodiments, the method is performed using protoplasts from: large area crops, for example, wheat, corn, soybean or cotton plants; high value or ornamental crops, for example, tomato, lettuce, pepper or pumpkin plants; turf or forage plants, for example, grass or alfalfa plants; or a test plant, e.g., an Arabidopsis plant or a snapdragon plant.

In certain embodiments, the pathogen is a plant pathogen or a molecule derived therefrom. The plant pathogen may be a virus, a bacterium, a fungal cell, or the like. In certain embodiments, the pathogen is a molecular agent (e.g., a viral capsid protein, flagellin, lipopolysaccharide, peptidoglycan, chitin protein) or a fragment thereof derived from a plant pathogen.

In another aspect, a kit for performing a method of identifying plant protoplasts that lack pathogen resistance is disclosed. The kit may include a microfluidic chip and reagents for detecting viability of plant protoplasts. The microfluidic chip may have a configuration according to any of the microfluidic chips disclosed herein. For example, the microfluidic chip may include a housing having a flow region and at least one growth chamber, and optionally, at least one surface of the growth chamber may include a surface-modifying ligand or a covalently-linked coating material. The reagent for detecting the viability of plant protoplasts can be a fluorescent stain, e.g., Fluorescein Diacetate (FDA), Hoechst, a fluorescent whitening agent, a chlorophyll stain, and the like.

These and other features and advantages of the disclosed methods and kits will be set forth or will become more fully apparent in the description that follows and in the appended claims. The features and advantages may be realized and obtained by means of the instruments and combinations particularly pointed out in the appended examples, partial list of embodiments and claims. Furthermore, the features and advantages of the described methods may be learned by the practice or will be obvious from the description, as set forth hereinafter.

Drawings

Fig. 1A illustrates a microfluidic device and a system with associated control apparatus according to some embodiments of the present disclosure.

Fig. 1B illustrates a microfluidic device with an isolation pen according to an embodiment of the present disclosure.

Fig. 2A-2B illustrate a microfluidic device with isolation pens according to some embodiments of the present disclosure.

Fig. 2C illustrates an isolation pen of a microfluidic device according to some embodiments of the present disclosure.

Fig. 3 illustrates an isolation pen of a microfluidic device according to some embodiments of the present disclosure.

Fig. 4A-4B illustrate electrokinetic features of a microfluidic device according to some embodiments of the present disclosure.

Fig. 5A illustrates a system for use with a microfluidic device and associated control apparatus according to some embodiments of the present disclosure.

Fig. 5B illustrates an imaging device according to some embodiments of the present disclosure.

Fig. 6 is an example of one embodiment of a process for perfusing a fluidic medium in a microfluidic device.

Fig. 7 is an example of another embodiment of a process of perfusing a fluidic medium in a microfluidic device.

FIG. 8 depicts a photographic representation of grape protoplasts cultured according to one embodiment of the methods described herein.

FIG. 9 depicts a photographic representation of lettuce protoplasts cultured according to one embodiment of the methods described herein.

FIG. 10 is a schematic of a method of genotyping a plant protoplast according to the methods described herein.

FIG. 11 is a schematic representation of a method of identifying a disease resistance trait according to the methods described herein.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

This specification describes exemplary embodiments and applications of the disclosure. However, the present disclosure is not limited to these exemplary embodiments and applications, nor to the manner in which the exemplary embodiments and applications operate or are described herein. Further, the figures may show simplified or partial views, and the sizes of elements in the figures may be exaggerated or not in proportion. In addition, as the terms "on," "attached to," "connected to," "coupled to" or similar words are used herein, an element (e.g., a material, a layer, a substrate, etc.) may be "on," "attached to," "connected to" or "coupled to" another element, whether the element is directly on, attached to, connected to or coupled to the other element, or one or more intervening elements may be present between the element and the other element. Additionally, unless the context dictates otherwise, directions (e.g., above, below, top, bottom, side, up, down, directly below, directly above, below, horizontal, vertical, "x", "y", "z", etc.), if provided, are provided by way of example only and for ease of illustration and discussion and not by way of limitation. Where a list of elements (e.g., elements a, b, c) is referred to, such reference is intended to include any one of the listed elements themselves, any combination of fewer than all of the listed elements, and/or combinations of all of the listed elements. The division of the sections in the specification is for ease of review only and does not limit any combination of the elements described.

Where the dimensions of a microfluidic feature are described as having a width or area, the dimensions are generally described with respect to x-axis and/or y-axis dimensions, both lying in a plane parallel to the substrate and/or lid of the microfluidic device. The height of the microfluidic features can be described with respect to a z-axis direction that is perpendicular to a plane parallel to a substrate and/or a cover of the microfluidic device. In some cases, the cross-sectional area of a microfluidic feature (such as a channel or passageway) may be referenced to the x-axis/z-axis, y-axis/z-axis, or x-axis/y-axis area.

As used herein, "substantially" means sufficient for the intended purpose. The term "substantially" thus allows for minor, non-obvious variations from absolute or perfect states, dimensions, measurements, results, etc., such as would be expected by one of ordinary skill in the art but without significantly affecting overall performance. "substantially" when used in relation to a numerical value or a parameter or characteristic that may be represented as a numerical value is within ten percent.

The term "plurality" means more than one.

The term "plurality" as used herein may be 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

As used herein, μm refers to microns, μm3 refers to cubic microns, pL refers to picoliters, nL refers to nanoliters, and μ L (or uL) refers to microliters.

The term "disposed" as used herein includes within its meaning "located".

As used herein, a "microfluidic device" or "microfluidic apparatus" is a device comprising one or more discrete microfluidic circuits configured to hold a fluid, each microfluidic circuit comprising fluidically interconnected circuit elements (including, but not limited to, regions, flow paths, channels, chambers, and/or pens), and at least one port configured to allow a fluid (and optionally, micro-objects suspended in the fluid) to flow into and/or out of the microfluidic device. Typically, the microfluidic circuit of a microfluidic device will include a flow region (which may include or be a microfluidic channel) and at least one chamber, and will accommodate a volume of less than about 1mL (e.g., less than about 750, 500, 250, 200, 150, 100, 75, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, or 2 μ L) of fluid. In some embodiments, the microfluidic circuit maintains about 1-2, 1-3, 1-4, 1-5, 2-8, 2-10, 2-12, 2-15, 2-20, 5-30, 5-40, 5-50, 10-75, 10-100, 20-150, 20-200, 50-250, or 50-300 μ L. The microfluidic circuit may be configured to have a first end in fluid communication with a first port (e.g., an inlet) in the microfluidic device and a second end in fluid communication with a second port (e.g., an outlet) in the microfluidic device.

As used herein, a "nanofluidic device" or "nanofluidic apparatus" is a type of microfluidic device having a microfluidic circuit that includes at least one circuit element configured to accommodate a volume of fluid of less than about 1 μ L (e.g., less than about 750, 500, 250, 200, 150, 100, 75, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1nL or less). The nanofluidic device can include a plurality of circuit elements (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10,000, or more). In certain embodiments, one or more (e.g., all) of the at least one loop element is configured to accommodate a volume of fluid of about 100pL to 1nL, 100pL to 2nL, 100pL to 5nL, 250pL to 2nL, 250pL to 5nL, 250pL to 10nL, 500pL to 5nL, 500pL to 10nL, 500pL to 15nL, 750pL to 10nL, 750pL to 15nL, 750pL to 20nL, 1 to 10nL, 1 to 15nL, 1 to 20nL, 1 to 25nL, or 1 to 50 nL. In other embodiments, one or more (e.g., all) of the at least one circuit element is configured to hold a volume of fluid of about 20nL to 200nL, 100 to 300nL, 100 to 400nL, 100 to 500nL, 200 to 300nL, 200 to 400nL, 200 to 500nL, 200 to 600nL, 200 to 700nL, 250 to 400nL, 250 to 500nL, 250 to 600nL, or 250 to 750 nL.

Microfluidic devices may be referred to herein as "microfluidic chips" or "chips," and nanofluidic devices may be referred to herein as "nanofluidic chips" or "chips.

As used herein, "microfluidic channel" or "flow channel" refers to a flow region of a microfluidic device that is significantly longer than both the horizontal and vertical dimensions. For example, the flow channel may be at least 5 times the length of the horizontal or vertical dimension, such as at least 10 times the length, at least 25 times the length, at least 100 times the length, at least 200 times the length, at least 500 times the length, at least 1,000 times the length, at least 5,000 times the length, or more. In some embodiments, the length of the flow channel is about 100,000 microns to about 500,000 microns, including any value between 100,000 microns and 500,000 microns. In some embodiments, the horizontal dimension is about 100 microns to about 1000 microns (e.g., about 150 microns to about 500 microns) and the vertical dimension is about 25 microns to about 200 microns (e.g., about 40 microns to about 150 microns). It should be noted that the flow channels may have a variety of different spatial configurations in the microfluidic device and are therefore not limited to entirely linear elements. For example, the flow channel may be or include one or more segments having the following configuration: curved, tortuous, spiral, inclined, descending, diverging (e.g., multiple distinct flow paths), and any combination thereof. In addition, the flow channel may have different cross-sectional areas along its path, widening and narrowing to provide the desired fluid flow therein. The flow channel may comprise a valve, and the valve may be of any type known in the art of microfluidics. Examples of microfluidic channels including valves are disclosed in U.S. Pat. Nos. 6,408,878(Unger et al) and 9,227,200(Chiou et al), both of which are incorporated herein by reference in their entirety.

As used herein with respect to a fluid medium, "diffusion" refers to the thermodynamic movement of a component of the fluid medium down a concentration gradient.

The phrase "flow of the medium" means bulk movement of the fluid medium primarily due to any mechanism other than diffusion. For example, the flow of the medium may involve the fluid medium moving from one point to another due to a pressure difference between the points. Such flow may include continuous, fluctuating, periodic, random, intermittent, or reciprocating flow of liquid, or any combination thereof. When one fluid medium flows into another fluid medium, turbulence and mixing of the media may result.

The phrase "substantially non-flowing" refers to a flow rate of the fluid medium that has an average rate over time that is less than a diffusion rate of a component of the material (e.g., the analyte of interest) into or within the fluid medium. The diffusion rate of the components of such materials may depend on, for example, the temperature, the size of the components, and the strength of the interaction between the components and the fluid medium.

The phrase "in fluid communication" as used herein with respect to different regions within a microfluidic device refers to the fluid communication in each region to form a single body of fluid when the different regions are substantially filled with a fluid, such as a fluidic medium. This does not mean that the fluids (or fluid media) in the different regions must be identical in composition. In contrast, fluids in different fluidly-connected regions of a microfluidic device may have different compositions (e.g., different concentrations of solutes, such as proteins, carbohydrates, ions, or other molecules) that vary as the solutes move along their respective concentration gradients and/or the fluids flow through the microfluidic device.

As used herein, a "flow path" refers to one or more fluidly communicating circuit elements (e.g., channels, regions, chambers, etc.) that define and experience a trajectory of a flow of a medium. Thus, the flow path is an example of a swept area (swept region) of a microfluidic device. Other circuit elements (e.g., unswept areas) may be in fluid communication with the circuit elements comprising the flow path without experiencing the flow of the medium in the flow path.

As used herein, an "isolated microobject" constitutes a defined region that confines the microobject within the microfluidic device.

As used herein, an "isolation region" refers to a region within a microfluidic device that is configured to receive a micro-object such that the micro-object is not drawn away from the region by fluid flow through the microfluidic device. Depending on the context, the term "isolation region" may also refer to a structure defining the region, which may include a base/substrate, a wall (e.g., made of microfluidic circuit material), and a lid.

Microfluidic (or nanofluidic) devices may include "swept" areas and "unswept" areas. As used herein, a "swept" area includes one or more fluidically interconnected circuit elements of a microfluidic circuit, each fluidically interconnected circuit element experiencing a flow of a medium as fluid flows through the microfluidic circuit. The circuit elements of the swept area may include, for example, areas, channels, and all or part of the chamber. As used herein, an "unswept" area includes one or more fluidically interconnected circuit elements of a microfluidic circuit, each of which circuit elements experiences substantially no flow of fluid as it flows through the microfluidic circuit. The unswept region may be fluidly connected to the swept region, provided that the fluid communication is configured to enable diffusion but substantially no media flow between the swept region and the unswept region. Thus, the microfluidic device may be configured to substantially isolate the non-swept area from the flow of the medium in the swept area, while such that there is substantially only diffusive fluid communication between the swept area and the non-swept area. For example, the flow channels of a microfluidic device are examples of swept areas, while isolated areas of a microfluidic device (described in further detail below) are examples of unswept areas.

The term "light transmissive" as used herein refers to a material that allows visible light to pass through without substantially changing the light as it passes through.

As used herein, "bright field" illumination and/or images refer to white light illumination from a microfluidic field of view of a broad spectrum light source, wherein contrast is created by absorption of light by objects in the field of view.

As used herein, "structured light" is projected light that illuminates a portion of the surface of a device without illuminating an adjacent portion of the surface. Structured light is typically generated by a structured light modulator (e.g., a Digital Mirror Device (DMD), a micro-shutter array system (MSA), a Liquid Crystal Display (LCD), etc.). Structured light can be corrected for surface irregularities as well as for irregularities associated with the light projection itself, e.g. image dropout (fall-off) at the edge of the illumination field.

As used herein, the "clear aperture" of a lens (or lens assembly) is the diameter or size of the portion of the lens (or lens assembly) that may be used for its intended purpose. In some cases, the clear aperture may be substantially equal to the physical diameter of the lens (or lens assembly). However, due to manufacturing limitations, it may be difficult to create a clear aperture equal to the actual physical diameter of the lens (or lens assembly).

The term "active area" as used herein refers to the portion of an image sensor or structured light modulator that may be used to image or provide structured light to a field of view in a particular optical device, respectively. The active area is constrained by an optical device, such as an aperture stop for the optical path within the optical device. Although the active area corresponds to a two-dimensional surface, the measurement result of the active area generally corresponds to the length of a diagonal line passing through opposite corners of a square having the same area.

As used herein, an "image beam" is an electromagnetic wave that is reflected or emitted from a surface of a device, micro-object, or fluid medium that is being viewed by an optical apparatus. The device may be any of the microfluidic devices described herein. The micro-object and the fluid medium may be located within such a microfluidic device.

The term "micro-object" as used herein generally refers to any microscopic object that can be isolated and/or manipulated according to the present disclosure. Non-limiting examples of micro-objects include: inanimate micro-objects, e.g. microparticles, microbeads (e.g. polystyrene beads, Luminex)^TMBeads, etc.), magnetic beads, nanorods, microwires, quantum dots, etc.; biological micro-objects, such as cells, biological organelles, vesicles or recombinants, synthetic vesicles, liposomes (e.g., synthetic or derived from membrane preparations), lipid nanorafts, and the like; or a combination of inanimate micro-objects and biological micro-objects (e.g., microbeads attached to cells, liposome-coated microbeads, liposome-coated magnetic beads, etc.). The beads may include covalently or non-covalently attached moieties/molecules, such as fluorescent labels, nucleic acids (e.g., oligonucleotides), proteins, carbohydrates, antigens, small molecule signaling moieties, or other chemical/biological species that can be used in an assay. Lipid nanorafts have been described in, for example, Ritchieet al (2009) "stabilization of Membrane Proteins in Phospholipid bilayers Nanodiscs," Methods enzymol, 464: 211 and 231.

The term "cell" as used herein is used interchangeably with the term "biological cell". Non-limiting examples of biological cells include eukaryotic cells, plant cells, animal cells (e.g., mammalian cells, reptile cells, avian cells, fish cells, etc.); prokaryotic cells, bacterial cells, fungal cells, protozoan cells, and the like; cells dissociated from tissues, such as muscle, cartilage, fat, skin, liver, lung cells, nerve, glial cells, and the like; immune cells such as T cells, B cells, plasma cells, natural killer cells, macrophages, and the like; embryos (e.g., zygotes), germ cells, such as oocytes, OVA, sperm cells, and the like; fused cells, hybridomas, cultured cells, cells from cell lines, cancer cells, infected cells, transfected and/or transformed cells, reporter cells, and the like. Mammalian cells can be, for example, from humans, mice, rats, horses, goats, sheep, cattle, pigs, primates, and the like.

A colony of biological cells is a "clone" if all living cells in the colony that can be propagated are daughter cells derived from a single parent cell. In certain embodiments, all daughter cells in the clonal colony are derived from no more than 10 divisions of a single parent cell. In other embodiments, all daughter cells in a clonal colony are derived from no more than 14 divisions of a single parental cell. In other embodiments, all daughter cells in the clonal colony are derived from no more than 17 divisions of a single parent cell. In other embodiments, all daughter cells in the clonal colony are derived from no more than 20 divisions of a single parent cell. The term "clonal cells" refers to cells of the same clonal colony.

As used herein, a "colony" of biological cells refers to 2 or more cells (e.g., about 2 to about 20, about 4 to about 40, about 6 to about 60, about 8 to about 80, about 10 to about 100, about 20 to about 200, about 40 to about 400, about 60 to about 600, about 80 to about 800, about 100 to about 1000, or greater than 1000 cells).

As used herein, the term "maintaining cells" refers to providing an environment comprising fluid and gas components and optionally surfaces that provide the conditions necessary to keep the cells viable and/or expanded.

The term "expansion" as used herein in reference to a cell refers to an increase in the number of cells.

A "component" of a fluid medium is any chemical or biochemical molecule present in the medium, including solvent molecules, ions, small molecules, antibiotics, nucleotides and nucleosides, nucleic acids, amino acids, peptides, proteins, sugars, carbohydrates, lipids, fatty acids, cholesterol, metabolites, and the like.

As used herein, a "capture moiety" is a chemical or biological species, function or motif that provides a site of identification for a micro-object. The selected class of micro-objects can identify the capture moiety generated in situ and can bind or have an affinity for the capture moiety generated in situ. Non-limiting examples include antigens, antibodies, and cell surface binding motifs.

"antibody" as used herein refers to immunoglobulin (Ig) and includes polyclonal and monoclonal antibodies; multi-chain antibodies, such as IgG, IgM, IgA, IgE, and IgD antibodies; single chain antibodies, such as camelid antibodies; mammalian antibodies, including primate antibodies (e.g., human), rodent antibodies (e.g., mouse, rat, guinea pig, hamster, etc.), lagomorph antibodies (e.g., rabbit), ungulate antibodies (e.g., bovine, porcine, equine, donkey, camel, etc.), and canine antibodies (e.g., dog); primatized (e.g., humanized) antibodies; chimeric antibodies, e.g., mouse-human, mouse-primate antibodies, and the like; and can be an intact molecule or a fragment thereof (e.g., a light chain variable region (VL), a heavy chain variable region (VH), scFv, Fv, Fd, Fab ', and F (ab') 2 fragment), or a multimer or aggregate of intact molecules and/or fragments; and may occur in nature or be produced, for example, by immunization, synthesis, or genetic engineering. The term "antibody fragment" as used herein refers to a fragment derived from or associated with an antibody, which binds an antigen. In some embodiments, antibody fragments may be derivatized to exhibit structural features that promote clearance and absorption, for example, by the addition of galactose residues. The ability of a biological micro-object (e.g., a biological cell) to produce a particular biological material (e.g., a protein, such as an antibody) can be determined in such a microfluidic device. In particular embodiments of assays, sample material containing biological micro-objects (e.g., cells) to be assayed for production of an analyte of interest may be loaded into a swept area of a microfluidic device. Some of the biological micro-objects (e.g., mammalian cells, such as human cells) may be selected for a particular characteristic and disposed in an unswept area. The remaining sample material may then flow out of the swept area and may be the analyte material flowing into the swept area. Because the selected biological micro-objects are in the unswept area, the selected biological micro-objects are substantially unaffected by the flow of the remaining sample material or the flow of the assay material. The selected biological micro-objects may be allowed to produce analytes of interest that may diffuse from the unswept area into the swept area, where the analytes of interest may react with the analyte material to produce locally detectable reactions, each of which may be associated with a particular unswept area. Any unswept areas associated with the detected reactions can be analyzed to determine which, if any, of the biological micro-objects in the unswept areas are sufficient producers of the analyte of interest.

An antigen as referred to herein is a molecule or portion thereof that can specifically bind to another molecule, such as an antigen-specific receptor. An antigen can be any part of a molecule, such as a conformational epitope or a linear molecular fragment, and can be generally identified by a highly variable antigen receptor of the adaptive immune system (B cell receptor or T cell receptor). The antigen may comprise a peptide, polysaccharide or lipid. An antigen may be characterized by its ability to bind to the variable Fab region of an antibody. Different antibodies have the potential to distinguish between different epitopes present on the surface of an antigen, the structure of which can be modulated by the presence of a hapten, which can be a small molecule.

The ability of a biological micro-object (e.g., a biological cell) to produce a particular biological material (e.g., a protein, such as an antibody) can be determined in a microfluidic device. In particular embodiments of assays, sample material containing biological micro-objects (e.g., cells) to be assayed for production of an analyte of interest may be loaded into a swept area of a microfluidic device. Some of the biological micro-objects (e.g., plant cells, such as plant protoplasts) can be selected for a particular characteristic and disposed in an unswept area. The remaining sample material may then flow out of the swept area and may be the analyte material flowing into the swept area. Because the selected biological micro-objects are in the unswept area, the selected biological micro-objects are substantially unaffected by the flow of the remaining sample material or the flow of the assay material. The selected biological micro-objects may be allowed to produce analytes of interest that may diffuse from the unswept area into the swept area, where the analytes of interest may react with the analyte material to produce locally detectable reactions, each of which may be associated with a particular unswept area. Any unswept areas associated with the detected reactions can be analyzed to determine which, if any, of the biological micro-objects in the unswept areas are sufficient producers of the analyte of interest.

Microfluidic device/system feature cross-applicability. It should be understood that various features of the microfluidic devices, systems, and kinetic techniques described herein may be combinable or interchangeable. For example, the features described herein with reference to microfluidic devices 100, 175, 200, 300, 320, 400, 450, 520 and system attributes as described in fig. 1A-5B may be combinable or interchangeable.

A microfluidic device. Fig. 1A shows an example of a microfluidic device 100. A perspective view of the microfluidic device 100 is shown with a portion of the cover 110 cut away to provide a partial view into the microfluidic device 100. The microfluidic device 100 generally includes a microfluidic circuit 120, the microfluidic circuit 120 including a flow path 106 through which a fluidic medium 180 may flow, optionally carrying one or more micro-objects (not shown) into the microfluidic circuit 120 and/or through the microfluidic circuit 120.

As shown generally in fig. 1A, the microfluidic circuit 120 is defined by the housing 102. Although the housing 102 may be physically constructed in different configurations, in the example shown in fig. 1A, the housing 102 is depicted as including a support structure 104 (e.g., a base), a microfluidic circuit structure 108, and a cover 110. The support structure 104, the microfluidic circuit structure 108, and the cover 110 may be attached to one another. For example, the microfluidic circuit structure 108 may be disposed on an inner surface 109 of the support structure 104, and the cover 110 may be disposed over the microfluidic circuit structure 108. The microfluidic circuit structure 108 may define elements of the microfluidic circuit 120 with the support structure 104 and the lid 110, forming a three-layer structure.

As shown in fig. 1A, the support structure 104 may be located at the bottom of the microfluidic circuit 120 and the lid 110 may be located at the top of the microfluidic circuit 120. Alternatively, the support structure 104 and the cover 110 may be configured in other orientations. For example, the support structure 104 may be located at the top of the microfluidic circuit 120 and the lid 110 may be located at the bottom of the microfluidic circuit 120. In any event, there may be one or more ports 107, each of which includes a passage into or out of the housing 102. Examples of channels include valves, gates, through-holes, and the like. As shown, the port 107 is a through hole formed by a gap in the microfluidic circuit structure 108. However, the port 107 may be located in other components of the housing 102 (e.g., the cover 110). Only one port 107 is shown in fig. 1A, but the microfluidic circuit 120 may have two or more ports 107. For example, there may be a first port 107 that serves as an inlet for fluid into the microfluidic circuit 120 and a second port 107 that serves as an outlet for fluid out of the microfluidic circuit 120. Whether the port 107 serves as an inlet or an outlet may depend on the direction of fluid flow through the flow path 106.

The support structure 104 may include one or more electrodes (not shown) and a substrate (or a plurality of interconnected substrates). For example, the support structure 104 may include one or more semiconductor substrates, each semiconductor substrate electrically connected to an electrode (e.g., all or a subset of the semiconductor substrates may be electrically connected to a single electrode). The support structure 104 may also include a printed circuit board assembly ("PCBA"). For example, the semiconductor substrate may be mounted on a PCBA.

The microfluidic circuit structure 108 may define circuit elements of the microfluidic circuit 120. Such circuit elements may include spaces or regions that may be fluidically interconnected when the microfluidic circuit 120 is fluidically filled, such as flow regions (which may include or may be one or more flow channels), chambers (the class of circuit elements may also include subcategories that include exclusion fences), wells (traps), and the like. The loop element may also comprise a fence or the like. In the microfluidic circuit 120 shown in fig. 1A, the microfluidic circuit structure 108 includes a frame 114 and a microfluidic circuit material 116. The frame 114 may partially or completely surround the microfluidic circuit material 116. The frame 114 may be, for example, a relatively rigid structure that substantially surrounds the microfluidic circuit material 116. For example, the frame 114 may comprise a metallic material. However, the microfluidic circuit structure need not include a frame 114. For example, the microfluidic circuit structure may be composed of (or consist essentially of) microfluidic circuit material 116.

The microfluidic circuit material 116 can be patterned with chambers or the like to define circuit elements and interconnects of the microfluidic circuit 120, e.g., chambers, fences, and microfluidic channels. The microfluidic circuit material 116 may include a flexible material, such as a flexible polymer (e.g., rubber, plastic, elastomer, silicone, polydimethylsiloxane ("PDMS"), etc.), which may be gas permeable. Other examples of materials from which microfluidic circuit material 116 may be formed include molded glass, etchable materials such as silicone (e.g., photo-patternable silicone or "PPS"), photoresist (e.g., SU8), and the like. In some embodiments, such materials (i.e., microfluidic circuit material 116) may be rigid and/or substantially gas impermeable. Regardless, the microfluidic circuit material 116 may be disposed on the support structure 104 and within the frame 114.

The microfluidic circuit 120 may include a flow region in which one or more chambers may be disposed and/or with which one or more chambers may be in fluid communication. The chamber may have one or more openings that place it in fluid communication with one or more flow regions. In some embodiments, the flow region includes or corresponds to a microfluidic channel 122. Although a single microfluidic circuit 120 is shown in fig. 1A, a suitable microfluidic device may include a plurality (e.g., 2 or 3) of such microfluidic circuits. In some embodiments, the microfluidic device 100 may be configured as a nanofluidic device. As shown in fig. 1A, microfluidic circuit 120 can include a plurality of microfluidic isolation pens 124, 126, 128, and 130, where each isolation pen can have one or more openings. In some embodiments of an isolation pen, the isolation pen may have only a single opening in fluid communication with the flow path 106. In some other embodiments, the isolation pen may have more than one opening, e.g., n openings, in fluid communication with the flow path 106, but with n-1 valved openings such that all but one opening is closable. When all valved openings are closed, the isolation pen limits the exchange of material from the flow region to the isolation pen to only by diffusion. In some embodiments, the isolation pens include various features and structures (e.g., isolation regions) that are optimized to retain micro-objects within the isolation pens (and thus within a microfluidic device such as the microfluidic device 100) even as the medium 180 flows through the flow path 106.

The cover 110 may be an integral part of the frame 114 and/or the microfluidic circuit material 116. Alternatively, as shown in FIG. 1A, the cover 110 may be a structurally different element. The cover 110 may comprise the same or different material as the frame 114 and/or the microfluidic circuit material 116. In some embodiments, the cover 110 may be an integral part of the microfluidic circuit material 116. Similarly, the support structure 104 may be a separate structure from the frame 114 or microfluidic circuit material 116 as shown, or an integral part of the frame 114 or microfluidic circuit material 116. Likewise, the frame 114 and microfluidic circuit material 116 may be separate structures as shown in FIG. 1A or components of the same structure. Regardless of the various possible compositions, the microfluidic device may retain a three-layer structure including a base layer and a cover layer sandwiching an intermediate layer in which the microfluidic circuit 120 is located.

In some embodiments, the cover 110 may comprise a rigid material. The rigid material may be glass or a material with similar properties. In some embodiments, the cover 110 may comprise a deformable material. The deformable material may be a polymer, such as PDMS. In some embodiments, the cover 110 may comprise a rigid and deformable material. For example, one or more portions of the cover 110 (e.g., one or more portions located above the isolation rails 124, 126, 128, 130) can include a deformable material that interfaces with the rigid material of the cover 110. Microfluidic devices having a cover comprising rigid and deformable materials have been described, for example, in U.S. patent No. 10,058,865 (Breinlinger et al), the contents of which are incorporated herein by reference. In some embodiments, the cover 110 may further include one or more electrodes. One or more of the electrodes may comprise a conductive oxide, such as Indium Tin Oxide (ITO), which may be coated on glass or similar insulating material. Alternatively, the one or more electrodes may be flexible electrodes, e.g., single-walled nanotubes, multi-walled nanotubes, nanowires, clusters of conductive nanoparticles, or combinations thereof, embedded in a deformable material, such as a polymer (e.g., PDMS). Flexible electrodes that can be used in microfluidic devices have been described, for example, in U.S. patent No. 9,227,200 (Chiou et al), the contents of which are incorporated herein by reference. In some embodiments, the cover 110 and/or the support structure 104 are optically transparent. The cover 110 may also include at least one gas permeable material (e.g., PDMS or PPS).

In the example shown in fig. 1A, microfluidic circuit 120 is shown to include microfluidic channel 122 and isolation pens 124, 126, 128, 130. Each pen includes an opening to the channel 122, with the remainder being closed, such that the pen can substantially isolate micro-objects within the pen from the fluid medium 180 and/or micro-objects in the flow path 106 of the channel 122 or other pen. The walls of the isolation fence extend from the inner surface 109 of the base to the inner surface of the cover 110 for closure. The opening of the isolation pen to the microfluidic channel 122 is oriented at an angle to the flow path 106 of the fluidic medium 180 such that the flow 106 is not directed into the pen. The vector of bulk fluid flow in channel 122 can be tangent or parallel to the open plane of the isolation pen and is not directed into the opening of the pen. In some cases, the pens 124, 126, 128, 130 are configured to physically isolate one or more micro-objects within the microfluidic circuit 120. As will be discussed and illustrated in detail below, the isolation pens of the present disclosure may include various shapes, surfaces, and features optimized for use with DEP, OET, OEW, flowing fluids, magnetic forces, centripetal forces, and/or gravity.

Microfluidic circuit 120 may include any number of microfluidic isolation pens. Although five isolation pens are shown, microfluidic circuit 120 can have fewer or more isolation pens. As shown, each of the microfluidic isolation pens 124, 126, 128, and 130 of the microfluidic circuit 120 includes different features and shapes that can provide one or more benefits useful for maintaining, separating, assaying, or culturing biological micro-objects. In some embodiments, microfluidic circuit 120 includes a plurality of identical microfluidic isolation pens.

In the embodiment shown in FIG. 1A, a single flow path 106 is shown containing a single channel 122. However, other embodiments may include multiple channels 122 within a single flow path 106, as shown in FIG. 1B. Microfluidic circuit 120 also includes an inlet valve or port 107 in fluid communication with flow path 106, and fluid medium 180 may enter flow path 106 (and channel 122) through inlet valve or port 107. In some cases, the flow path 106 comprises a substantially straight path. In other cases, the flow path 106 is arranged in a non-linear or tortuous manner (e.g., a zigzag pattern), whereby the flow path 106 travels over the microfluidic device 100 two or more times, e.g., in alternating directions. The flow in the flow path 106 may travel from the inlet to the outlet, or may reverse direction and travel from the outlet to the inlet.

One example of a multi-channel device (microfluidic device 175) that is otherwise similar to microfluidic device 100 is shown in fig. 1B. Microfluidic device 175 and its constituent circuit elements (e.g., channel 122 and isolation pen 128) can have any of the dimensions discussed herein. The microfluidic circuit shown in fig. 1B has two inlet/outlet ports 107 and a flow path 106 containing four different channels 122. The number of channels into which the microfluidic circuit is subdivided may be selected to reduce fluidic resistance. For example, a microfluidic circuit may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more channels to provide a selected range of fluidic resistances. Microfluidic device 175 also includes a plurality of isolation pens open at each channel 122, wherein each isolation pen is similar to isolation pen 128 of fig. 1A and can have any size or function of any isolation pen as described herein. However, the isolation pens of microfluidic device 175 can have different shapes, such as isolation pens 124, 126, or 130 of fig. 1A or any shape as described elsewhere herein. Furthermore, microfluidic device 175 can include mixed isolation pens with different shapes. In some cases, a plurality of isolation pens are configured (e.g., relative to channel 122) such that the isolation pens can be loaded with target micro-objects in parallel.

Returning to fig. 1A, the microfluidic circuit 120 may also include one or more optional micro-object wells (traps) 132. An optional well 132 may be formed in the wall bounding the channel 122 and may be positioned opposite the opening of one or more of the microfluidic isolation pens 124, 126, 128, 130. The optional trap 132 may be configured to receive or capture a single micro-object from the flow path 106, or may be configured to receive or capture multiple micro-objects from the flow path 106. In some cases, optional well 132 comprises a volume approximately equal to the volume of a single target micro-object.

Isolating the fence. The microfluidic devices described herein can include one or more isolation pens, wherein each isolation pen is adapted to hold one or more micro-objects (e.g., biological cells or groups of cells associated together). The isolation pens can be disposed within and open to a flow region, which in some embodiments is a microfluidic channel. Each isolation pen may have one or more openings for fluid communication with one or more microfluidic channels. In some embodiments, the isolation pen may have only one opening for the microfluidic channel.

Fig. 2A-2C illustrate isolation pens 224, 226, and 228 of microfluidic device 200, which can be similar to isolation pen 128 of fig. 1A. Each isolation pen 224, 226, and 228 can include an isolation region 240 and a communication region 236, the communication region 236 fluidly connecting the isolation region 240 to a flow region, which in some embodiments can include a microfluidic channel (e.g., channel 122). The communication region 236 may include a proximal opening 234 to the flow region (e.g., microfluidic channel 122) and a distal opening 238 to an isolation region 240. The communication zone 236 can be configured such that a maximum penetration depth of flow of fluidic media (not shown) flowing in the fluid channel 122 through the isolation pens 224, 226, and 228 does not extend into the isolation zone 240, as discussed below with respect to fig. 2C. In some embodiments, streamlines from flow in the microfluidic channel do not enter the isolation region. Thus, due to the communication zone 236, micro-objects (not shown) or other materials (not shown) disposed in the isolation zones 240 of the isolation pens 224, 226, and 228 can be isolated from the flow of the fluidic medium 180 in the fluidic channel 122 and substantially unaffected by the flow of the fluidic medium 180.

Isolation pens 224, 226, and 228 of fig. 2A-2C each have a single opening directly into microfluidic channel 122. As shown in fig. 2A, the opening of the isolation pen can open laterally from the microfluidic channel 122, fig. 2A depicts a vertical cross-section of the microfluidic device 200. Fig. 2B shows a horizontal cross-section of the microfluidic device 200. Electrode activation substrate 206 can be located underneath both microfluidic channel 122 and isolation pens 224, 226, and 228. The upper surface of the electrode activation substrate 206 within the housing of the isolation pen forming the bottom surface of the isolation pen may be disposed at the same height or substantially the same height as the upper surface of the electrode activation substrate 206 within the microfluidic channel 122 (or flow region if a channel is not present) forming the bottom surface of the flow channel (or flow region) of the microfluidic device. The electrode activation substrate 206 may be featureless or may have an irregular or patterned surface with depressions that vary from its highest height to its lowest height by less than about 3 microns, 2.5 microns, 2 microns, 1.5 microns, 1 micron, 0.9 microns, 0.5 microns, 0.4 microns, 0.2 microns, 0.1 microns, or less. The variation in height of the upper surface of the substrate over the microfluidic channel 122 (or flow region) and the isolation fence can be equal to or less than about 10%, 7%, 5%, 3%, 2%, 1%, 0.9%, 0.8%, 0.5%, 0.3%, or 0.1% of the height of the isolation fence wall. Alternatively, the height variation of the upper surface of the substrate over the microfluidic channel 122 (or flow region) and the isolation pens may be equal to or less than about 2%, 1%, 0.9%, 0.8%, 0.5%, 0.3%, 0.2%, or 0.1% of the height of the substrate. Although the microfluidic device 200 is described in detail, this may also apply to any of the microfluidic devices described herein.

Microfluidic channel 122 and communication region 236 can be examples of swept regions, while isolation region 240 of isolation pens 224, 226, and 228 can be examples of unswept regions. Isolation pens like 224, 226, 228, etc. have isolation zones, where each isolation zone has only one opening that leads to a communicating zone of the isolation pen. The exchange of fluid medium into and out of the thus configured isolation region may be limited to occur substantially only by diffusion. As noted, microfluidic channel 122 and isolation pens 224, 226, and 228 can be configured to hold one or more fluidic media 180, as described above. In the example shown in fig. 2A-2B, port 222 is connected to microfluidic channel 122 and allows fluidic medium 180 to be introduced into microfluidic device 200 or removed from microfluidic device 230. Prior to introduction of fluid medium 180, the microfluidic device may be filled with a gas, such as carbon dioxide gas. Once the microfluidic device 200 contains fluidic medium 180, a flow 242 of fluidic medium 180 in the microfluidic channel 122 may be selectively created and stopped (see fig. 2C). For example, as shown, the ports 222 may be disposed at different locations (e.g., opposite ends) of a flow region (e.g., microfluidic channel 122) and may cause a flow 242 of fluidic medium from one port 222 serving as an inlet to another port 222 serving as an outlet.

Fig. 2C illustrates a detailed view of an example of an isolation fence 224, where isolation fence 224 can contain one or more micro-objects 246, according to some embodiments. The flow 242 of fluidic medium 180 in microfluidic channel 122 through proximal opening 234 of communication region 236 of isolation fence 224 can cause an auxiliary flow 244 of fluidic medium 180 into and/or out of isolation fence 224. To separate micro-objects 246 in isolation region 240 of isolation fence 224 from auxiliary flow 244, length L of communication region 236 of isolation fence 224_con(i.e., from the proximal opening 234 to the distal opening 238) should be greater than the penetration depth D of the secondary flow 244 into the communication zone 236_p. Depth of penetration D_pDepending on a number of factors, including: the shape of the microfluidic channel 122, which may be defined by the width W of the communication region 236 at the proximal opening 234_conDefining; width W of microfluidic channel 122 at proximal opening 234_ch(ii) a Height H of channel 122 at proximal opening 234_ch(ii) a And the width of the distal opening 238 of the communication zone 236. Among these factors, the width W of the communication zone 236 at the proximal opening 234_conAnd the height H of the channel 122 at the proximal opening 234_chOften the most important. In addition, the penetration depth D_pMay be affected by the velocity of fluid medium 180 in passage 122 and the viscosity of fluid medium 180. However, these The factors (i.e., velocity and viscosity) may vary widely without significantly changing the penetration depth D_p. For example, for microfluidic chip 200, the width W of the communication region 236 at the proximal opening 234_conIs about 50 microns, and the height H of the channel 122 at the proximal opening 234_chIs about 40 microns, the width W of the microfluidic channel 122 at the proximal opening 234_chAbout 150 microns, depth of penetration D of the secondary flow 244_pLess than W from a flow rate of 0.1. mu.l/sec_con1.0 times (i.e., less than 50 microns) to a flow rate of 20 microliters/sec_con2.0 times (i.e., about 100 microns), which represents a 200-fold increase relative to the velocity of the fluid medium 180, D_pOnly 2.5 times.

In some embodiments, the walls of microfluidic channel 122 and isolation pens 224, 226, or 228 may be oriented with respect to the vector of flow 242 of fluidic medium 180 in microfluidic channel 122 as follows: microfluidic channel width W_ch(or cross-section of the microfluidic channel 122) may be substantially perpendicular to the flow 242 of the medium 180; width W of communication region 236 at opening 234_con(or cross-sectional plane) may be substantially parallel to the flow 242 of the medium 180 in the microfluidic channel 122; and/or length L of the communicating region_conMay be substantially perpendicular to the flow 242 of the medium 180 in the microfluidic channel 122. The foregoing are merely examples, the relative positions of microfluidic channel 122 and isolation pens 224, 226, and 228 may be in different orientations relative to one another.

In some embodiments, the configuration of the microfluidic channel 122 and the opening 234 may be fixed for a given microfluidic device, while the rate of flow 242 of the fluidic medium 180 in the microfluidic channel 122 may be variable. Thus, for each isolation fence 224, the maximum velocity V of flow 242 of fluid medium 180 in channel 122 can be identified_maxWhich ensures the penetration depth D of the secondary flow 244_pNot exceeding the length L of the communicating region 236_con. When not exceeding V_maxWhen desired, the generated secondary flow 244 may be contained entirely within the communication zone 236 and not into the isolation zone 240. Thus, preventing flow 242 of fluidic medium 180 in microfluidic channel 122 (swept area) would beMicro-object 246 is pulled out of isolation region 240 (which is an unswept region of the microfluidic circuit), resulting in micro-object 246 being retained in isolation region 240. Thus, selection of microfluidic circuit element dimensions and further selection of operating parameters (e.g., velocity of fluid medium 180) can prevent material from microfluidic channel 122 or another isolation fence 226 or 228 from contaminating isolation region 240 of isolation fence 224. It should be noted, however, that for many microfluidic chip configurations, there is no need to worry about V _maxIn itself, because V is being implemented_maxPreviously, the chip would have ruptured from the pressure associated with the high velocity flow of fluid medium 180.

The components (not shown) in the first fluidic medium 180 in the microfluidic channel 122 can mix with the second fluidic medium 248 in the isolation region 240 substantially only in the following manner: components of the first medium 180 diffuse from the microfluidic channel 122 through the communication region 236 and into the second fluid medium 248 in the isolation region 240. Similarly, the components (not shown) of the second medium 248 in the isolation region 240 may mix with the first medium 180 in the microfluidic channel 122 substantially only by: the constituents of the second media 248 diffuse from the isolation zone 240, through the communication zone 236, and into the first media 180 in the microfluidic channel 122. In some embodiments, the degree of fluid medium exchange by diffusion between the isolation region and the flow region of the isolation pen is greater than about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or greater than about 99% of the fluid exchange.

In some embodiments, the first media 180 may be the same media as the second media 248 or a different media. In some embodiments, the first medium 180 and the second medium 248 may be initially the same and then become different (e.g., by modulation of the second medium 248 by one or more cells in the isolation region 240, or by altering the medium 180 flowing through the microfluidic channel 122).

As shown in FIG. 2C, the width W of the communicating region 236_conMay be uniform from the proximal opening 234 to the distal opening 238. Width W of communication zone 236 at distal opening 238_conMay be open proximally of the communication zone 236, hereinWidth W at 234_conAny value identified. In some embodiments, the width of the isolation region 240 at the distal opening 238 may be the same as the width W of the communication region 236 at the proximal opening 234_conAre substantially the same. Alternatively, the width of the communication zone 236 at the distal opening 238 may be different (e.g., greater or less) than the width W of the communication zone 236 at the proximal opening 234_con. In some embodiments, the width W of the communication zone 236_conMay narrow or widen between the proximal opening 234 and the distal opening 238. For example, the communication zone 236 may narrow or widen between the proximal and distal openings using a variety of different geometries (e.g., chamfered communication zone). Further, any portion or sub-portion of the communication region 236 (e.g., a portion of the communication region adjacent the proximal opening 234) may narrow or widen.

Fig. 3 depicts another exemplary embodiment of a microfluidic device 300 including a microfluidic circuit structure 308. Microfluidic device 300 includes channel 322 and isolation pen 324. Isolation fence 324 has similar features and characteristics as described herein for any of the isolation fences of microfluidic devices 100, 175, 200, 400, 520 and any other microfluidic devices described herein.

The exemplary microfluidic device of fig. 3 includes a microfluidic channel 322 having a width W as described herein_chAnd contains a flow 310 of first fluid medium 302 and one or more isolation pens 324 (shown only in fig. 3). The isolation fences 324 each have a length L_sA communication region 336 and an isolation region 340, wherein the isolation region 340 contains the second fluid medium 304. The communication region 336 has a width W_con1And a proximal opening 334 and width W to the microfluidic channel 322_con2And to the distal opening 338 of the isolation region 340. Width W as described herein_{con 1}Can be reacted with W_{con 2}The same or different. The walls of each isolation fence 324 can be formed from microfluidic circuit material 316, and microfluidic circuit material 316 can also form walls 330 of the communication region. The wall 330 of the communication zone may correspond to being positioned laterally with respect to the proximal opening 334 and extending at least partiallyInto the enclosed portion of isolation fence 324. In some embodiments, the length L of the connected region 336_conAt least partially by the length L of the wall 330 of the communication zone_wallAnd (4) limiting. The wall 330 of the communication zone may have a penetration depth D selected to be greater than the secondary flow 344_pLength L of_wall. Thus, the secondary flow 344 may be contained entirely within the communication region without extending into the isolation region 340.

The wall 330 of the communication zone can define a hooked region 352, the hooked region 352 being a sub-region of the isolation region 340 of the isolation fence 324. Since the wall 330 of the communicating region extends into the interior chamber of the isolation fence, at the selected length L_wallIn this case, the walls 330 of the communication zone may act as a physical barrier to shield the hook-shaped region 352 from the auxiliary flow 344, constituting a hook-shaped region. In some embodiments, the length L of the wall 330 of the communication region_wallThe longer the hooked region 352 is, the more occluded.

In an isolation pen configured similar to those of fig. 2A-2C and 3, the isolation region can have any type of shape and size, and can be selected to regulate diffusion of nutrients, reagents, and/or media into the isolation pen to reach a distal wall of the isolation pen, e.g., opposite a proximal opening of the communication region to the flow region (or microfluidic channel). The size and shape of the isolation zone can be further selected to regulate the diffusion of waste products and/or secretion products of the biological micro-objects from the isolation zone to the flow zone via the proximal opening of the communicating region of the isolation pen. In general, the shape of the isolation region is not critical to the ability of the isolation pen to isolate the micro-object from direct flow in the flow region.

In some other embodiments of the isolation pen, the isolation region can have more than one opening that fluidly communicates the isolation region with a flow region of the microfluidic device. However, for an isolation zone having n number of openings fluidly connecting the isolation zone to the flow zone (or two or more flow zones), n-1 openings may be valved. When closing the n-1 valved openings, the isolation zone has only one effective opening, so the exchange of material into and out of the isolation zone is done only by diffusion.

Examples of microfluidic devices having pens in which biological micro-objects can be placed, cultured and/or monitored have been described in, for example, the following documents: U.S. patent No. 9,857,333 (Chapman et al), U.S. patent No. 10,010,882 (White et al), and U.S. patent No. 9,889,445 (Chapman et al), each of which is incorporated herein by reference in its entirety.

Insulating fence dimensions. As described herein, various dimensions and/or characteristics of the isolation pens and the microfluidic channels to which the isolation pens open can be selected to limit the introduction of contaminants or unwanted micro-objects from the flow region/microfluidic channel into the isolation region of the isolation pens; limiting the exchange of components in the fluid medium from the channel or from the isolation zone to substantially diffusion exchange; facilitating the transfer of micro-objects into and out of the isolation fence; and/or promoting the growth or expansion of biological cells. For any of the embodiments described herein, the microfluidic channels and the isolation pens can have any suitable combination of dimensions, which can be selected by one of skill in the art in light of the teachings of the present disclosure below.

The proximal opening of the communicating region of the isolation fence can have a width (e.g., W)_conOr_Wcon1): the width is at least as large as the largest dimension of the micro-object (e.g., biological cell, which may be a plant cell (e.g., plant protoplast)) for which the isolation pen is intended. In some embodiments, the proximal opening has a width (e.g., W) of about 20 microns, about 40 microns, about 50 microns, about 60 microns, about 75 microns, about 100 microns, about 150 microns, about 200 microns, or about 300 microns_conOr_Wcon1). The foregoing are examples only, the width of the proximal opening (e.g., W)_conOr_Wcon1) May be selected to be a value between any of the values listed above (e.g., about 20-200 microns, about 20-150 microns, 20-100 microns, about 20-75 microns, about 20-60 microns, about 50-300 microns, about 50-200 microns, about 50-150 microns, about 50-100 microns, about 50-75 microns, about 75-150 microns, about 75-100 microns, about 100-300 microns, about 100-200 microns, or about 200-300 microns).

In some embodiments, the communicating region of the isolation fence can have a length (e.g., L) from the proximal opening to the distal opening to the isolation region of the isolation fence_con) Which is the width of the proximal opening (e.g., W) _conOr_Wcon1) At least 0.5 times, at least 0.6 times, at least 0.7 times, at least 0.8 times, at least 0.9 times, at least 1.0 times, at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.75 times, at least 2.0 times, at least 2.25 times, at least 2.5 times, at least 2.75 times, at least 3.0 times, at least 3.5 times, at least 4.0 times, at least 4.5 times, at least 5.0 times, at least 6.0 times, at least 7.0 times, at least 8.0 times, at least 9.0 times, or at least 10.0 times of (b). Thus, for example, the proximal opening of the communicating region of the isolation pen can have a width (e.g., W) of about 20 microns to about 200 microns (e.g., about 50 microns to about 150 microns)_conOr W_con1) And the length L of the communicating region_conMay be at least 1.0 times (e.g., at least 1.5 times or at least 2.0 times) the width of the proximal opening. As another example, the proximal opening of the communication region of the isolation fence can have a width (e.g., W) of about 20 microns to about 100 microns (e.g., about 20 microns to about 60 microns)_conOr W_con1) And the length L of the communicating region_conMay be at least 1.0 times (e.g., at least 1.5 times or at least 2.0 times) the width of the proximal opening.

The microfluidic channel of the microfluidic device to which the isolation fence leads can have a specified dimension (e.g., width or height). In some embodiments, the height of the microfluidic channel at the proximal opening to the communication region of the isolation pen (e.g., H) _ch) May be in any of the following ranges: 20-100 microns, 20-90 microns, 20-80 microns, 20-70 microns, 20-60 microns, 20-50 microns, 30-100 microns, 30-90 microns, 30-80 microns, 30-70 microns, 30-60 microns, 30-50 microns, 40-100 microns, 40-90 microns, 40-80 microns, 40-70 microns, 40-60 microns, or 40-50 microns. The above are examples only, and the height (e.g., H) of the microfluidic channel (e.g., 122) is_ch) May be selected to be between any of the values listed above. In addition, exclusion in microfluidic channelsHeight of microfluidic channel 122 (e.g., H) in a region other than at the proximal opening of the pen_ch) May be selected to be at any of these heights.

Width of microfluidic channel at proximal opening to communication region of isolation fence (e.g., W)_ch) May be in any of the following ranges: about 20-500 microns, 20-400 microns, 20-300 microns, 20-200 microns, 20-150 microns, 20-100 microns, 20-80 microns, 20-60 microns, 30-400 microns, 30-300 microns, 30-200 microns, 30-150 microns, 30-100 microns, 30-80 microns, 30-60 microns, 40-300 microns, 40-200 microns, 40-150 microns, 40-100 microns, 40-80 microns, 40-60 microns, 50-1000 microns, 50-500 microns, 50-400 microns, 50-300 microns, 50-250 microns, 50-200 microns, 50-150 microns, 50-100 microns, 50-80 microns, 60-300 microns, 60-200 microns, 60-150 microns, 60-100 microns, 60-80 microns, 70-500 microns, 70-400 microns, 70-300 microns, 70-250 microns, 70-200 microns, 70-150 microns, 70-100 microns, 80-100 microns, 90-400 microns, 90-300 microns, 90-250 microns, 90-200 microns, 90-150 microns, 100-300 microns, 100-250 microns, 100-200 microns, 100-150 microns, 100-120 microns, 200-800 microns, 200-700 microns or 200-600 microns. The foregoing are examples only, the width of the microfluidic channel (e.g., W) _ch) May be selected to be a value between any of the values listed above. Also, the width (e.g., W) of the microfluidic channel 122 in a region of the microfluidic channel other than the proximal opening of the isolation fence_ch) May be selected to be any of these widths.

The cross-sectional area of the microfluidic channel at the proximal opening of the communication region leading to the isolation rail may be about 500-, 3,000 and 7,500 square microns or 3,000 to 6,000 square microns. The foregoing are merely examples and the cross-sectional area of the microfluidic channel at the proximal opening may be selected to be between any of the values listed above. In various embodiments, the cross-sectional area of the microfluidic channel at a region other than at the proximal opening may also be selected to be between any of the values listed above. In some embodiments, the cross-sectional area is selected to be a substantially uniform value for the entire length of the microfluidic channel.

In some embodiments, the microfluidic chip is configured such that the proximal opening (e.g., 234 or 334) of the communication region of the isolation fence can have a width (e.g., W) of about 20 microns to about 200 microns (e.g., about 50 microns to about 150 microns)_conOr W_con1) The communication region may have a length L that is at least 1.0 times (e.g., at least 1.5 times or at least 2.0 times) the width of the proximal opening_con(e.g., 236 or 336) and the microfluidic channel can have a height (e.g., H) at the proximal opening of about 30 microns to about 60 microns_ch). As another example, the proximal opening (e.g., 234 or 334) of the communication region of the isolation fence can have a width (e.g., W) of about 20 microns to about 100 microns (e.g., about 20 microns to about 60 microns)_conOr W_con1) The communication region may have a length L that is at least 1.0 times (e.g., at least 1.5 times or at least 2.0 times) the width of the proximal opening_con(e.g., 236 or 336) and the microfluidic channel can have a height (e.g., H) at the proximal opening of about 30 microns to about 60 microns_ch). The foregoing are examples only, and the width (e.g., W) of the proximal opening (e.g., 234 or 274)_conOr W_con1) Length of the connected region (e.g., L)_con) And/or width (e.g., W) of microfluidic channel (e.g., 122 or 322) _ch) May be a value selected to be between any of the values listed above.

In some embodiments, the proximal opening of the communicating region of the isolation pen (e.g.,234 or 334) may have a height (e.g., H) that is the flow region/microfluidic channel at the proximal opening_ch) A width (e.g., W) of 2.0 times or less (e.g., 2.0 times, 1.9 times, 1.8 times, 1.5 times, 1.3 times, 1.0 times, 0.8 times, 0.5 times, or 0.1 times) of_conOr W_con1) Or have a value within a range defined by any two of the above values.

In some embodiments, the width W of the proximal opening (e.g., 234 or 334) of the communicating region of the isolation fence_con1Width W of a distal opening (e.g., 238 or 338) that can be in communication with an isolation region of an isolation fence_con2The same is true. In some embodiments, the width W of the proximal opening_con1May be different from the width W of the distal opening_con2And W is_con1And/or W_con2Can be selected from the group consisting of_conOr W_con1Any of the values described. In some embodiments, the walls defining the proximal and distal openings (including the walls of the communication region) may be substantially parallel with respect to each other. In some embodiments, the walls defining the proximal and distal openings may be selected to be non-parallel with respect to each other.

The length (e.g., L) of the connected region_con) May be about 1-600 microns, 5-550 microns, 10-500 microns, 15-400 microns, 20-300 microns, 20-500 microns, 40-400 microns, 60-300 microns, 80-200 microns, about 100-150 microns, about 20-300 microns, about 20-250 microns, about 20-200 microns, about 20-150 microns, about 20-100 microns, about 30-250 microns, about 30-200 microns, about 30-150 microns, about 30-100 microns, about 30-80 microns, about 30-50 microns, about 45-250 microns, about 45-200 microns, about 45-100 microns, about 45-80 microns, about 45-60 microns, about 60-200 microns, about 60-150 microns, about 60-100 microns, or about 60-80 microns. The foregoing are examples only, the length of the communication region (e.g., L)_con) May be selected to be a value between any of the values listed above.

The length of the wall of the communicating region of the isolation fence (e.g., L)_wall) The width of the proximal opening (e.g., W) that can be the communicating region of the isolation fence_conOr W_con1) At least 0.5 times, at least 0.6 times, at least0.7 times, at least 0.8 times, at least 0.9 times, at least 1.0 times, at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.75 times, at least 2.0 times, at least 2.25 times, at least 2.5 times, at least 2.75 times, at least 3.0 times, or at least 3.5 times. In some embodiments, the walls of the connected regions can have a length L of about 20-200 microns, about 20-150 microns, about 20-100 microns, about 20-80 microns, or about 20-50 microns _wall. The foregoing are merely examples, and the walls of the connected regions may have a length L selected to be between any of the values listed above_wall。

The isolation fence can have a length L of about 40-600 microns, about 40-500 microns, about 40-400 microns, about 40-300 microns, about 40-200 microns, about 40-100 microns, or about 40-80 microns_s. The foregoing are merely examples, and an isolation fence may have a length L selected to be between any of the values listed above_s。

According to some embodiments, the isolation fence may have a specified height (e.g., H)_s). In some embodiments, the isolation fence has a height H of about 20 microns to about 200 microns (e.g., about 20 microns to about 150 microns, about 20 microns to about 100 microns, about 20 microns to about 60 microns, about 30 microns to about 150 microns, about 30 microns to about 100 microns, about 30 microns to about 60 microns, about 40 microns to about 150 microns, about 40 microns to about 100 microns, or about 40 microns to about 60 microns)_s. The foregoing are merely examples, and an isolation fence can have a height H selected to be between any of the values listed above_s。

Height H of the communicating region at the proximal opening of the isolation fence_conMay be any one of the following heights: 20-100 microns, 20-90 microns, 20-80 microns, 20-70 microns, 20-60 microns, 20-50 microns, 30-100 microns, 30-90 microns, 30-80 microns, 30-70 microns, 30-60 microns, 30-50 microns, 40-100 microns, 40-90 microns, 40-80 microns, 40-70 microns, 40-60 microns, or 40-50 microns. The foregoing are examples only, and the height H of the communication zone _conMay be selected to be between any of the values listed above. Typically, the height H of the communicating region_conIs selected to be in communication with the microfluidic channelHeight H at the proximal opening of (a)_chThe same is true. In addition, the height H of the isolation fence_sIs generally selected to be the height H of the communicating region_conAnd/or height H of the microfluidic channel_chThe same is true. In some embodiments, H_s、H_conAnd H_chMay be selected to be the same as any of the values listed above for the selected microfluidic device.

The isolation region may be configured to accommodate only one, two, three, four, five, or a similar relatively small number of micro-objects. In other embodiments, the isolation region may accommodate more than 10, more than 50, or more than 100 micro-objects. Thus, the volume of the isolation region may be, for example, at least 1x10⁴、1x10⁵、5x10⁵、8x10⁵、1x10⁶、2x10⁶、4x10⁶、6x10⁶、1x10⁷、3x10⁷、5x10⁷、1x10⁸、5x10⁸Or 8x10⁸Cubic microns or larger. The foregoing are merely examples, the isolation region may be configured to accommodate various numbers of micro-objects, and the volume is selected to be between any of the values listed above (e.g., at 1x 10)⁵Cubic micron sum of 5x10⁵Volume between cubic microns, 5x10⁵Cubic micron sum of 1x10⁶Volume between cubic microns, 1x10⁶Cubic micron sum 2x10⁶Volume between cubic microns, or medium 2x0 ⁶Cubic micron sum of 1x0⁷Volume between cubic microns).

According to some embodiments, the isolation pens of the microfluidic device can have a specified volume. The specified volume of the isolation pen (or isolation region of the isolation pen) can be selected such that a single cell or a small number (e.g., 2-10 or 2-5) of cells can rapidly condition the medium to obtain favorable (or optimal) growth conditions. In some embodiments, the isolation fence has about 5x10⁵、6x10⁵、8x10⁵、1x10⁶、2x10⁶、4x10⁶、8x10⁶、1x10⁷、3x10⁷、5x10⁷Or about 8x10⁷Cubic micrometers or greater.In some other embodiments, the isolation pen has a volume of about 1 nanoliter to about 50 nanoliters, 2 nanoliters to about 25 nanoliters, 2 nanoliters to about 20 nanoliters, about 2 nanoliters to about 15 nanoliters, or about 2 nanoliters to about 10 nanoliters. The foregoing are merely examples, and an isolation fence may have a volume selected to be any value between any of the values listed above.

According to some embodiments, the flow of fluidic media (e.g., 122 or 322) within the microfluidic channel may have a specified maximum velocity (e.g., V)_max). In some embodiments, the maximum speed (e.g., V)_max) Can be set at about 0.2, 0.5, 0.7, 1.0, 1.3, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.7, 7.0, 7.5, 8.0, 8.5, 9.0, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 microliters/second. The foregoing are merely examples, and the flow of fluidic media within a microfluidic channel may have a maximum velocity (e.g., V) selected to be a value between any of the values listed above _max)。

In various embodiments, the microfluidic device has an isolation pen configured as in any of the embodiments discussed herein, wherein the microfluidic device has from about 5 to about 10 isolation pens, from about 10 to about 50 isolation pens, from about 25 to about 200 isolation pens, from about 100 to about 500 isolation pens; about 200 to about 1000 insulation pens, about 500 to about 1500 insulation pens, about 1000 to about 2500 insulation pens, about 2000 to about 5000 insulation pens, about 3500 to about 7000 insulation pens, about 5000 to about 10000 insulation pens, about 7,500 to about 20,000 insulation pens, about 12,500 to about 25,000 insulation pens, about 20,000 to about 30,000 insulation pens, about 25,000 to about 35,000 insulation pens, about 30,000 to about 40,000 insulation pens, about 35,000 to about 45,000 insulation pens, or about 40,000 to about 50,000 insulation pens. The isolation pens need not all be the same size and can include multiple configurations (e.g., different widths, different features within the isolation pens).

Coating solution and coating agent. In some embodiments, at least one interior surface of the microfluidic device comprises a coating material that provides a layer suitable for maintaining, amplifying, and/or moving a biological micro-object (i.e., the biological micro-object exhibits increased viability, greater amplification, and/or greater portability within the microfluidic device). The conditioned surface can reduce surface fouling, participate in providing a hydration layer, and/or otherwise protect biological micro-objects from contact with non-organic materials inside the microfluidic device.

In some embodiments, substantially all of the interior surfaces of the microfluidic device comprise a coating material. The coated interior surface can include a surface of a flow region (e.g., a channel), a chamber, or an isolation pen, or a combination thereof. In some embodiments, each of the plurality of isolation pens has at least one interior surface coated with a coating material. In other embodiments, each of the plurality of flow regions or channels has at least one inner surface coated with a coating material. In some embodiments, at least one inner surface of each of the plurality of isolation pens and each of the plurality of channels is coated with a coating material. The coating may be applied before or after introduction of the biological micro-objects, or may be introduced simultaneously with the biological micro-objects. In some embodiments, the biological micro-objects may be input into the microfluidic device in a fluid medium comprising one or more coating agents. In other embodiments, the interior surface of a microfluidic device (e.g., a microfluidic device having an electrode-activated substrate, such as, but not limited to, a device including Dielectrophoresis (DEP) electrodes) can be treated or "primed" with a coating solution comprising a coating agent prior to introduction of biological micro-objects into the microfluidic device. Any convenient coating agent/coating solution may be used, including but not limited to: serum or serum factors, Bovine Serum Albumin (BSA), polymers, detergents, enzymes, and any combination thereof.

Coating materials based on synthetic polymers. At least one of the inner surfaces may include a coating material comprising a polymer. The polymer may be non-covalently bound (e.g., it may non-specifically adhere) to at least one surface. The polymer can have a variety of structural motifs, for example, all of the structural motifs found in block (and copolymers), star (star copolymers), and graft or comb (graft copolymers), all of which can be adapted for use in the methods disclosed herein. Multiple kinds ofAlkylene ether-containing polymers may be suitable for use in the microfluidic devices described herein, including but not limited to such asL44, L64, P85 and F127 (including F127NF), etcA polymer. Other examples of suitable coating materials are described in US2016/0312165, the contents of which are incorporated herein by reference in their entirety.

A covalently linked coating material. In some embodiments, at least one interior surface includes covalently attached molecules that provide a layer of organic and/or hydrophilic molecules suitable for maintaining/amplifying biological micro-objects within the microfluidic device. The covalently linked molecules include a linking group, wherein the linking group is covalently linked to one or more surfaces of the microfluidic device, as described below. The linking group is also linked to a surface modifying moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for maintaining/amplifying a biological micro-object.

In some embodiments, a covalently linked moiety configured to provide an organic and/or hydrophilic molecular layer suitable for maintaining/amplifying a microbial organism may comprise an alkyl or fluoroalkyl (including perfluoroalkyl) moiety; monosaccharides or polysaccharides (which may include but are not limited to dextran); alcohols (including but not limited to propargyl alcohol); polyols, including but not limited to polyvinyl alcohol; alkylene ethers including, but not limited to, polyethylene glycol; polyelectrolytes (including but not limited to polyacrylic acid or polyvinylphosphonic acid); amino (including derivatives thereof such as, but not limited to, alkylated amines, hydroxyalkylated amino, guanidino, and heterocyclic groups containing nitrogen ring atoms that are not aromatic, such as, but not limited to morpholinyl or piperazinyl); carboxylic acids, including but not limited to propiolic acid (which can provide a carboxylate anionic surface); phosphonic acids, including but not limited to ethynylphosphonic acid (which can provide a phosphonate anionic surface); a sulfonate anion; a carboxybetaine; a sulfobetaine; sulfamic acid; or an amino acid.

In various embodiments, a covalently linked moiety configured to provide an organic and/or hydrophilic molecular layer suitable for maintaining/amplifying a biological micro-object in a microfluidic device may comprise a non-polymeric moiety, such as an alkyl moiety, an amino acid moiety, an alcohol moiety, an amino moiety, a carboxylic acid moiety, a phosphonic acid moiety, a sulfonic acid moiety, an aminosulfonic acid moiety, or a sugar moiety. Alternatively, the covalently linked moieties may comprise polymeric moieties, which may comprise any of these moieties.

In some embodiments, the microfluidic device may have a hydrophobic layer on an inner surface of the base, the hydrophobic layer comprising covalently linked alkyl moieties. The covalently attached alkyl moiety can include carbon atoms that form a straight chain (e.g., a straight chain of at least 10 carbons or at least 14, 16, 18, 20, 22 or more carbons) and can be an unbranched alkyl moiety. In some embodiments, the alkyl group may include a substituted alkyl group (e.g., some carbons in the alkyl group may be fluorinated or perfluorinated). In some embodiments, the alkyl group can include a first segment that is linked to a second segment, the first segment can include a perfluoroalkyl group, and the second segment can include an unsubstituted alkyl group, wherein the first segment and the second segment can be linked directly or indirectly (e.g., by way of an ether linkage). The first segment of the alkyl group can be located distal to the linking group and the second segment of the alkyl group can be located proximal to the linking group.

In other embodiments, the covalent linking moiety can include at least one amino acid, which can include more than one type of amino acid. Thus, the covalent linking moiety may comprise a peptide or a protein. In some embodiments, the covalent linking moiety may comprise an amino acid, which may provide a zwitterionic surface to support cell growth, viability, portability, or any combination thereof.

In other embodiments, the covalently linked moiety may also include a streptavidin or biotin moiety. In some embodiments, a modified biological moiety (e.g., a biotinylated protein or peptide) may be introduced to the interior surface of a microfluidic device bearing covalently linked streptavidin and coupled to the surface via the covalently linked streptavidin, thereby providing a modified surface that presents the protein or peptide.

In other embodiments, the covalent linking moiety can comprise at least one alkylene oxideAnd may include any of the alkylene oxide polymers described above. One useful class of alkylene ether-containing polymers is polyethylene glycol (PEG M)_w<100,000Da) or polyethylene oxide (PEO, M)_w>100,000). In some embodiments, the PEG can have an M of about 1000Da, 5000Da, 10,000Da, or 20,000Da_w. In some embodiments, the PEG polymer may also be substituted with hydrophilic or charged moieties, such as, but not limited to, alcohol functionality (alcohol functionality) or carboxylic acid moieties.

The covalent linking moiety may comprise one or more sugars. The covalently linked sugar may be a monosaccharide, disaccharide or polysaccharide. Covalently linked sugars can be modified to introduce reactive partner moieties that allow coupling or refinement for attachment to a surface. An exemplary covalently linked moiety may include dextran polysaccharide, which may be indirectly coupled to a surface through an unbranched linker (linker).

The coating material providing the conditioned surface may comprise only one type of covalent linking moiety, or may comprise more than one different type of covalent linking moiety. For example, a polyethylene glycol-modified surface can have a covalently attached alkylene oxide moiety having a specified number of all the same alkylene oxide units, e.g., having the same linking group and covalent attachment to the surface, the same total length, and the same number of alkylene oxide units. Alternatively, the coating material may have more than one covalent attachment moiety attached to the surface. For example, the coating material may include a coating material having covalently attached alkylene oxide moieties (molecules having a specified number of alkylene oxide units, and may also include another set of molecules having, for example, a large to partial portion of a protein or peptide attached to a covalently attached alkylene oxide linking moiety having a greater number of alkylene oxide units. the different types of molecules may be varied in any suitable ratio to achieve the desired surface characteristics. for example, a conditioned surface having a mixture of first molecules having a chemical structure with a first specified number of alkylene oxide units and second molecules having a peptide or protein moiety, and a second molecule having a chemical structure with a first specified number of alkylene oxide units and a second molecule having a peptide or protein moiety, the peptide or protein moiety may be coupled to a covalently attached alkylene linking moiety via a biotin/streptavidin binding pair. In this case, the first set of molecules with distinct, less sterically demanding ends and fewer main chain atoms may help functionalize the entire substrate surface, preventing undesired adhesion or contact with the silicon/silicon oxide, hafnium oxide or aluminum oxide constituting the substrate itself. The choice of the mixture ratio of the first molecule to the second molecule may also modulate the surface modification introduced by the second molecule bearing the peptide or protein moiety.

Conditioned surface properties. Various factors can alter the physical thickness of the conditioned surface, such as the manner in which the conditioned surface is formed on the substrate (e.g., vapor deposition, liquid deposition, spin coating, dipping, and electrostatic coating). In some embodiments, the conditioned surface may have a thickness of about 1nm to about 10 nm. In some embodiments, the covalently linked portions of the conditioned surface can form a monolayer when covalently linked to the surface of a microfluidic device (which can include an electrode-activated substrate with Dielectrophoresis (DEP) or Electrowetting (EW) electrodes), and can have a thickness of less than 10nm (e.g., less than 5nm, or about 1.5 to 3.0 nm). These values are in sharp contrast to the thickness of the surface prepared by spin coating, which may typically have a thickness of about 30 nm. In some embodiments, the conditioned surface does not require a perfectly formed monolayer to be suitable for operation within a DEP configured microfluidic device. In other embodiments, the conditioned surface formed by covalently linked moieties may have a thickness of about 10nm to about 50 nm.

Single or multi-part conditioned surfaces. The covalently linked coating material may be formed by reaction of molecules already containing moieties configured to provide an organic and/or hydrophilic molecular layer suitable for maintaining/amplifying a biological micro-object in a microfluidic device, and may have the structure of formula I, as shown below. Alternatively, the covalently linked coating material may be formed as a two-part sequence by coupling a part configured to provide a layer of organic and/or hydrophilic molecules suitable for maintaining and/or amplifying biological micro-objects to a surface-modifying ligand which itself has been covalently linked to the surface, and has the structure of formula II. In some embodiments, the surface may be formed in a two-part or three-part sequence, including a streptavidin/biotin binding pair, to introduce proteins, peptides, or mixed modified surfaces.

The coating material can be covalently attached to an oxide of the surface of the substrate in either the DEP configuration or the EW configuration. The coating material may be attached to the oxide through a linking group ("LG"), which may be a siloxy or phosphonate group formed from the reaction of a siloxane or phosphonate group with the oxide. The portion configured to provide a layer of organic and/or hydrophilic molecules suitable for maintaining/amplifying a biological micro-object in a microfluidic device may be any portion described herein. The linking group LG may be directly or indirectly attached to a moiety configured to provide an organic and/or hydrophilic molecular layer suitable for maintaining/amplifying a biological micro-object in a microfluidic device. When the linking group LG is directly connected to the moiety, there is no optional linker ("L") and n is 0. When the linking group LG is indirectly linked to the moiety, a linker L is present and n is 1. The linker L may have a linear portion, wherein the backbone of the linear portion may include 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and/or phosphorus atoms, which are limited by chemical bonding, as is known in the art. It may be interrupted by any combination of one or more moieties selected from ether, amino, carbonyl, amido or phosphonate groups, arylene, heteroarylene and/or heterocyclic groups. In some embodiments, the coupling group CG represents the resulting group from the reaction of the reactive moiety Rx and the reactive partner moiety Rpx (i.e., the moiety configured to react with the reactive moiety Rx). CG may be a carboxamide group, a triazolylene group, a substituted triazolylene group, a carboxamide group, a thioamide group, an oxime group, a mercapto group, a disulfide group, an ether or an alkenyl group, or any other suitable group that may be formed upon reaction of a reactive moiety with its respective reactive partner. In some embodiments, CG may also represent a streptavidin/biotin binding pair.

Further details of suitable coating treatments and modifications and methods of preparation may be found in U.S. patent application publication No. US2016/0312165(Lowe, jr. et al), U.S. patent application publication No. US2017/0173580 (Lowe, jr. et al), international patent application publication No. WO2017/205830 (Lowe, jr. et al), and international patent application publication No. WO2019/01880 (beemuller et al), the disclosures of each of which are incorporated herein by reference in their entirety.

Microfluidic device dynamics. The microfluidic devices described herein may be used with any type of kinetic technique. As described herein, the control and monitoring apparatus of the system may include a power module for selecting and moving objects, such as micro-objects or droplets, in the microfluidic circuit of the microfluidic device. The kinetic techniques may include, for example, Dielectrophoresis (DEP), Electrowetting (EW), and/or other kinetic techniques. Microfluidic devices may have a variety of dynamic configurations depending on the type of object to be moved and other considerations. Returning to fig. 1A, for example, the support structure 104 and/or the lid 110 of the microfluidic device 100 may include a DEP electrode activation substrate for selectively inducing a motive force on micro-objects in the fluidic medium 180 in the microfluidic circuit 120 to select, capture, and/or move individual micro-objects or groups of micro-objects.

In some embodiments, a motive force is applied to the fluidic medium 180 (e.g., in the flow path and/or in the isolation pen) via one or more electrodes (not shown) to manipulate, transport, separate, and sort the micro-objects located therein. For example, in some embodiments, a motive force is applied to one or more portions of the microfluidic circuit 120 in order to transfer a single micro-object from the flow path 106 into a desired microfluidic isolation pen. In some embodiments, the motive force is used to prevent micro-objects in the isolation pen from being dislodged therefrom. Further, in some embodiments, a motive force is used to selectively remove previously collected micro-objects from the isolation fence according to embodiments of the present disclosure.

In some embodiments, the microfluidic device is configured as an optically actuated electrokinetic device, for example in an optoelectronic tweezers (OET) and/or an optical-electrowetting (OEW) configured device. Examples of suitable OET configured devices (e.g., comprising an optically actuated dielectrophoretic electrode activation substrate) may include those described in: U.S. patent No. RE 44,711 (Wu et al) (originally disclosed as U.S. patent No. 7,612,355), U.S. patent No. 7,956,339 (Ohta et al), U.S. patent No. 9,908,115 (Hobbs et al), and U.S. patent No. 9,403,172 (Short et al), each of which is incorporated herein by reference in its entirety. Examples of suitable OEW configured devices may include those described in the following documents: U.S. patent No. 6,958,132 (Chiou et al) and U.S. patent application No. 9,533,306 (Chiou et al), each of which is incorporated herein by reference in its entirety. Examples of suitable optically actuated electrically powered devices including combined OET/OEW configured devices may include those described in the following documents: U.S. patent application publication No. 2015/0306598 (Khandros et al), U.S. patent application publication No. 2015/0306599 (Khandros et al), and U.S. patent application publication No. 2017/0173580 (Lowe et al), each of which is incorporated herein by reference in its entirety.

It should be understood that the various examples of fig. 1-5B may show portions of a microfluidic device, while other portions are not depicted, for purposes of simplicity. 1-5B may be part of, and may be implemented as, one or more microfluidic systems. In one non-limiting example, fig. 4A and 4B show a side cross-sectional view and a top cross-sectional view, respectively, of a portion of the housing 102 of a microfluidic device 400 having a region/chamber 402, which region/chamber 402 can be part of a fluidic circuit element having a more detailed structure, such as a growth chamber, an isolation fence (which can be similar to any of the isolation fences described herein), a flow region, or a flow channel. For example, the microfluidic device 400 may be similar to the microfluidic devices 100, 175, 200, 300, 520 or any other microfluidic device described herein. Furthermore, the microfluidic device 400 may include other fluidic circuit elements, and may be part of a system including a control and monitoring apparatus 152 as described above, the control and monitoring apparatus 152 having one or more of a media module 160, a power module 162, an imaging module 164, an optional tilt module 166, and other modules 168. The microfluidics 175, 200, 300, 520, and any other microfluidic device described herein can similarly have any of the features described in detail with respect to fig. 1A-1B and 4A-4B.

As shown in the example of fig. 4A, the microfluidic device 400 includes a support structure 104 having a bottom electrode 404 and an electrode activation substrate 406 covering the bottom electrode 404, and a lid 110 having a top electrode 410, the top electrode 410 being spaced apart from the bottom electrode 404. The top electrode 410 and the electrode activation substrate 406 define opposing surfaces of the region/chamber 402. Thus, the fluid medium 180 contained in the region/chamber 402 provides a resistive connection between the top electrode 410 and the electrode activation substrate 406. Also shown is a power supply 412 configured to connect to the bottom electrode 404 and the top electrode 410 and to generate a bias voltage between the electrodes, which is required to generate the DEP force in the region/chamber 402. The power supply 412 may be, for example, an Alternating Current (AC) power supply.

In certain embodiments, the microfluidic device 200 shown in fig. 4A and 4B can have an optically actuated DEP electrode activation substrate. Thus, the changing pattern of light 418 from the light source 416 that can be controlled by the power module 162 can selectively activate and deactivate the changing pattern of DEP electrodes at the region 414 of the inner surface 408 of the electrode activation substrate 406. (hereinafter, the region 414 of the microfluidic device with DEP electrode activation substrate is referred to as the "DEP electrode region") as shown in fig. 4B, a light pattern 418 directed at the inner surface 408 of the electrode activation substrate 406 may illuminate a selected DEP electrode region 414a (shown in white) in a pattern such as a square. Hereinafter, DEP electrode regions 414 (hatched) that are not illuminated are referred to as "dark" DEP electrode regions 414. At each dark DEP electrode region 414, the relative electrical impedance through the DEP electrode activation substrate 406 (i.e., from the bottom electrode 404 up to the inner surface 408 of the electrode activation substrate 406 that interfaces with the fluid medium 180 in the flow region 106) is greater than the relative electrical impedance through the fluid medium 180 in the region/chamber 402 (i.e., from the inner surface 408 of the electrode activation substrate 406 to the top electrode 410 of the lid 110). However, at each irradiated DEP electrode region 414a, the irradiated DEP electrode region 414a exhibits a relatively reduced impedance through the electrode activation substrate 406 that is less than the relative impedance through the fluid medium 180 in the region/chamber 402.

Upon activation of the power supply 412, the aforementioned DEP configuration creates an electric field gradient in the fluidic medium 180 between the illuminated DEP electrode region 414a and the adjacent dark DEP electrode region 414, which in turn creates a local DEP force that attracts or repels nearby micro-objects (not shown) in the fluidic medium 180. Thus, DEP electrodes that attract or repel micro-objects in the fluidic medium 180 can be selectively activated and deactivated at many different such DEP electrode regions 414 at the inner surface 408 of the region/chamber 402 by varying the light pattern 418 projected from the light source 416 into the microfluidic device 400. Whether the DEP force attracts or repels nearby micro-objects may depend on parameters such as the frequency of the power source 412 and the dielectric properties of the fluid medium 180 and/or micro-objects (not shown). Depending on the frequency of the power applied to the DEP arrangement and the choice of fluid medium (e.g., a highly conductive medium such as PBS or other medium suitable for maintaining biological cells), a negative DEP force may be generated. Negative DEP forces may repel micro-objects away from the location of the induced non-uniform electric field. In some embodiments, microfluidic devices incorporating DEP technology can generate negative DEP forces.

The square pattern 420 of the illuminated DEP electrode region 414a shown in fig. 4B is merely an example. Any pattern of DEP electrode regions 414 can be illuminated (and thus activated) by a pattern of light 418 projected into the microfluidic device 400, and the pattern of illuminated/activated DEP electrode regions 414 can be repeatedly changed by changing or moving the light pattern 418.

In some embodiments, the electrode activation substrate 406 may include or consist of a photoconductive material. In such embodiments, the inner surface 408 of the electrode activation substrate 406 may be featureless. For example, the electrode activation substrate 406 may include or consist of a hydrogenated amorphous silicon (a-Si: H) layer. a-Si: h may comprise, for example, about 8% to 40% hydrogen (calculated as 100 hydrogen atoms/total number of hydrogen atoms and silicon atoms). a-Si: the H layer may have a thickness of about 500nm to about 2.0 μm. In such embodiments, DEP electrode regions 414 can be formed in any pattern anywhere on the inner surface 408 of the electrode activation substrate 406, according to the light pattern 418. Thus, the number and pattern of DEP electrode regions 214 need not be fixed, but may correspond to the light pattern 418. Examples of microfluidic devices having DEP configurations that include a photoconductive layer as described above have been described, for example, in U.S. patent RE44,711 (Wu et al), originally published as U.S. patent No. 7,612,355, the entire contents of which are incorporated herein by reference.

In other embodiments, electrode activation substrate 406 may comprise a substrate comprising a plurality of doped layers, electrically insulating layers (or regions), and conductive layers forming a semiconductor integrated circuit, such as is known in the semiconductor arts. For example, the electrode activation substrate 406 may include a plurality of phototransistors, including, for example, lateral bipolar phototransistors, wherein each phototransistor corresponds to a DEP electrode region 414. Alternatively, the electrode activation substrate 406 can include electrodes (e.g., conductive metal electrodes) controlled by phototransistor switches, each such electrode corresponding to a DEP electrode region 414. The electrode activation substrate 406 may include a pattern of such phototransistors or phototransistor control electrodes. For example, the pattern may be an array of substantially square phototransistor or phototransistor control electrodes arranged in rows and columns. Alternatively, the pattern may be an array of substantially hexagonal phototransistors or phototransistor control electrodes forming a hexagonal lattice. Regardless of the pattern, the return element can form electrical connections between the DEP electrode region 414 at the inner surface 408 of the electrode activation substrate 406 and the bottom electrode 404, and those electrical connections (i.e., phototransistors or electrodes) can be selectively activated and deactivated by the light pattern 418, as described above.

Examples of microfluidic devices having electrode-activated substrates comprising phototransistors have been described, for example, in the following documents: U.S. Pat. Nos. 7,956,339 (Ohta et al) and 9,908,115 (Hobbs et al), the entire contents of which are incorporated herein by reference. Examples of microfluidic devices having electrode-activated substrates with electrodes controlled by phototransistor switches have been described, for example, in U.S. patent No. 9,403,172 (Short et al), the entire contents of which are incorporated herein by reference.

In some embodiments of DEP configured microfluidic devices, the top electrode 410 is part of a first wall (or lid 110) of the housing 402, and the electrode activation substrate 406 and the bottom electrode 404 are part of a second wall (or support structure 104) of the housing 102. The region/chamber 402 may be located between the first wall and the second wall. In other embodiments, the electrode 410 is part of the second wall (or support structure 104) and one or both of the electrode activation substrate 406 and/or the electrode 410 is part of the first wall (or cover 110). Further, the light source 416 may alternatively be used to illuminate the housing 102 from below.

With the microfluidic device 400 of fig. 4A-4B having a DEP electrode activation substrate, the power module 162 of the control and monitoring apparatus 152 as described herein with respect to fig. 1A can select micro-objects (not shown) in the fluidic medium 180 in the region/chamber 402 by projecting a light pattern 418 into the microfluidic device 400 to activate a first set of one or more DEP electrodes at DEP electrode regions 414A of the inner surface 408 of the electrode activation substrate 406 in a pattern (e.g., a square pattern 420) that surrounds and captures the micro-objects. The motive module 162 may then move the in situ generated captured micro-objects by moving the light pattern 418 relative to the microfluidic device 400 to activate the second set of one or more DEP electrodes at the DEP electrode region 414. Alternatively, the microfluidic device 400 may be moved relative to the light pattern 418.

In other embodiments, the microfluidic device 400 may be a device that does not rely on a light activated DEP configuration of DEP electrodes at the inner surface 408 of the electrode activation substrate 406. For example, the electrode activation substrate 406 can include selectively addressable and energizable electrodes disposed opposite a surface (e.g., the cover 110) that includes at least one electrode. A switch (e.g., a transistor switch in a semiconductor substrate) can be selectively opened and closed to activate or deactivate the DEP electrode at the DEP electrode region 414, thereby creating a net DEP force on a micro-object (not shown) within the region/chamber 402 near the activated DEP electrode. Depending on such characteristics as the frequency of the power source 412 and the dielectric properties of the medium (not shown) and/or micro-objects in the region/chamber 402, the DEP force may attract or repel nearby micro-objects. By selectively activating and deactivating sets of DEP electrodes (e.g., at sets of DEP electrode regions 414 forming square pattern 420), one or more micro-objects in region/chamber 402 can be selected and moved within region/chamber 402. The power module 162 in fig. 1A can control such switches and thus activate and deactivate individual ones of the DEP electrodes to select and move particular micro-objects (not shown) surrounding the region/chamber 402. Microfluidic devices having DEP electrode activated substrates comprising selectively addressable and energizable electrodes are known in the art and have been described in U.S. patent No. 6,294,063 (Becker et al) and U.S. patent No. 6,942,776 (Medoro), each of which is incorporated herein by reference in its entirety.

Regardless of whether the microfluidic device 400 has a dielectrophoretic electrode activation substrate, an electrowetting electrode activation substrate, or a combination of both dielectrophoretic and electrowetting activation substrates, the power supply 412 may be used to provide a potential (e.g., an AC voltage potential) that powers the circuitry of the microfluidic device 400. The power supply 412 may be the same as or a component of the power supply 192 referenced in fig. 1A. The power supply 412 may be configured to provide an AC voltage and/or current to the top electrode 410 and the bottom electrode 404. For AC voltages, the power source 412 may provide a range of frequencies and a range of average or peak powers (e.g., voltages or currents) sufficient to generate a net DEP force (or electrowetting force) strong enough to select and move individual micro-objects (not shown) in the region/chamber 402, as described above, and/or to change the wetting properties of the inner surface 408 of the support structure 104 in the region/chamber 202, also as described above. Such frequency ranges and average or peak power ranges are known in the art. See, for example, U.S. patent No. 6,958,132 (Chiou et al), U.S. patent No. RE44,711 (Wu et al) (initially published as U.S. patent No. 7,612,355), and U.S. patent applications published as 2014/0124370(Short et al), 2015/0306598(Khandros et al), US2015/0306599(Khandros et al), and 2017/0173580(Lowe, jr. et al), the disclosures of each of which are incorporated herein by reference in their entirety.

Other forces may be utilized within the microfluidic device, alone or in combination, to move selected micro-objects. Bulk fluid flow within a microfluidic channel can move micro-objects within a flow region. Localized fluid flow, which can operate within a microfluidic channel, within an isolation pen, or within another chamber (e.g., a reservoir), can also be used to move selected micro-objects. The local fluid flow can be used to move selected micro-objects out of the flow region into a non-flow region (e.g., an isolation pen) or reverse operation, i.e., from the non-flow region into the flow region. The localized flow may be actuated by deforming a deformable wall of the microfluidic device, as described in U.S. patent No. 10,058,865 (Breinlinger et al), which is incorporated herein by reference in its entirety.

Gravity can be used to move micro-objects in a microfluidic channel into and/or out of an isolation pen or other chamber, as described in U.S. patent No. 9,744,533 (breinnlinger et al), which is incorporated herein by reference in its entirety. The use of gravity (e.g., by tilting the microfluidic device and/or a support to which the microfluidic device is attached) is useful for allowing a large amount of movement of cells from the flow region into the isolation pen or from the isolation pen into the flow region. Magnetic forces may be employed to move micro-objects comprising paramagnetic materials, which may include magnetic micro-objects attached to or associated with biological micro-objects. Alternatively or additionally, centripetal forces may be used to move micro-objects within a microfluidic channel, as well as to move them into or out of isolation pens or other chambers in a microfluidic device.

In another alternative to moving micro-objects, the laser generated moving force may be used to output micro-objects or to assist in outputting micro-objects from an isolation pen or any other chamber in a microfluidic device, as described in international patent publication No. WO2017/117408 (Kurz et al), which is incorporated herein by reference in its entirety.

In some embodiments, DEP forces are combined with other forces, such as fluid flow (e.g., bulk fluid flow in a channel or localized fluid flow actuated by deformation of a deformable surface of a microfluidic device, laser-generated movement forces, and/or gravity) in order to manipulate, transport, separate, and sort micro-objects and/or droplets within microfluidic circuit 120. In some embodiments, the DEP force may be applied before the other forces. In other embodiments, the DEP force may be applied after other forces. In other cases, the DEP force may be applied simultaneously with or alternating with other forces.

Provided is a system. Returning to fig. 1A, a system 150 for operating and controlling a microfluidic device (e.g., for controlling the microfluidic device 100) is shown. The power supply 192 may provide electrical power to the microfluidic device 100, providing a bias voltage or current as desired. The power supply 192 may, for example, include one or more Alternating Current (AC) and/or Direct Current (DC) voltage or current sources.

The system 150 may also include a media source 178. The media source 178 (e.g., container, reservoir, etc.) may include multiple portions or containers, each portion or container for holding a different fluid medium 180. Thus, as shown in fig. 1A, the media source 178 can be a device that is external to the microfluidic device 100 and separate from the microfluidic device 100. Alternatively, the media source 178 can be located wholly or partially within the housing 102 of the microfluidic device 100. For example, the media source 178 can include a reservoir that is part of the microfluidic device 100.

Fig. 1A also shows a simplified block diagram depiction of an example of a control and monitoring apparatus 152 that forms part of the system 150 and that may be used in conjunction with the microfluidic device 100. As shown, examples of such control and monitoring devices 152 include a master controller 154, including a media module 160 for controlling a media source 178, a power module 162 for controlling movement and/or selection of micro-objects (not shown) and/or media (e.g., droplets of media) in the microfluidic circuit 120, an imaging module 164 for controlling an imaging device (e.g., a camera, a microscope, a light source, or any combination thereof) for capturing images (e.g., digital images), and an optional tilt module 166 for controlling tilting of the microfluidic device 100. The control apparatus 152 may also include other modules 168 for controlling, monitoring, or performing other functions with respect to the microfluidic device 100. As shown, the monitoring device 152 may also include a display device 170 and an input/output device 172.

The master controller 154 may include a Control Module (CM)156 and a digital memory 158. The control module 156 may include, for example, a digital processor configured to operate in accordance with machine-executable instructions (e.g., software, firmware, source code, etc.) stored as non-transitory data or signals in the memory 158. Alternatively or additionally, the control module 156 may include hard-wired digital circuitry and/or analog circuitry. Media module 160, power module 162, imaging module 164, optional tilt module 166, and/or other modules 168 may be similarly configured. Thus, the functions, process actions, or process steps discussed herein as being performed with respect to the microfluidic device 100 or any other microfluidic device may be performed by any one or more of the master controller 154, the media module 160, the power module 162, the imaging module 164, the optional tilt module 166, and/or the other modules 168 configured as described above. Similarly, the master controller 154, the media module 160, the power module 162, the imaging module 164, the tilt module 166, and/or the other modules 168 may be communicatively coupled to send and receive data used in any of the functions, processes, actions, or steps discussed herein.

The media module 160 controls the media source 178. For example, the media module 160 may control the media source 178 to input a selected fluid media 180 into the housing 102 (e.g., through the inlet port 107). The media module 160 may also control the removal of media from the housing 102 (e.g., through an output port (not shown)). One or more media may thus be selectively input into the microfluidic circuit 120 or removed from the microfluidic circuit 120. The media module 160 may also control the flow of fluidic media 180 of the flow path 106 within the microfluidic circuit 120. The media module 160 may also provide conditioned gas conditions to the media source 178, for example, providing a gas containing 5% (or more) CO₂The environment of (2). The media module 160 may also control the temperature of the housing of the media source, for example, to provide appropriate temperature control for feeder cells in the media source.

And a power module. May be configured to control the selection and movement of micro-objects in the microfluidic circuit 120. The housing 102 of the microfluidic device 100 can include one or more electrokinetic mechanisms including a Dielectrophoresis (DEP) electrode activation substrate, an optoelectronic tweezers (OET) electrode activation substrate, an Electrowetting (EW) electrode activation substrate, and/or an optical-electrowetting (OEW) electrode activation substrate, wherein the power module 162 can control the activation of electrodes and/or transistors (e.g., phototransistors) to select and move micro-objects and/or droplets within the flow paths 106 and/or the isolation pens 124, 126, 128, and 130. The electrokinetic mechanism may be any suitable single or combined mechanism, as described in the paragraphs herein describing the kinetic techniques used in microfluidic devices. The DEP configured devices may include one or more electrodes that apply a non-uniform electric field in the microfluidic circuit 120 sufficient to exert dielectrophoretic forces on micro-objects in the microfluidic circuit 120. The OET configured apparatus may include photoactivatable electrodes for providing selective control of movement of micro-objects in the microfluidic circuit 120 via light-induced dielectrophoresis.

The imaging module 164 may control an imaging device. For example, the imaging module 164 may receive and process image data from an imaging device. The image data from the imaging device may include any type of information captured by the imaging device 194 (e.g., the presence or absence of micro-objects, droplets of media, accumulation of labels (such as fluorescent labels, etc.)). Using the information captured by the imaging device, the imaging module 164 may further calculate the location of objects (e.g., micro-objects, media drops) and/or the rate of movement of these objects within the microfluidic device 100.

The imaging device (part of the imaging module 164 discussed below) may include a device for capturing images within the microfluidic circuit 120, such as a digital camera. In some cases, the imaging device also includes a detector with a fast frame rate and/or high sensitivity (e.g., for low-light applications). The imaging device may also include a mechanism for directing the stimulated radiation and/or beams into the microfluidic circuit 120 and collecting radiation and/or beams reflected or emitted from the microfluidic circuit 120 (or micro-objects contained therein). The emitted light beam may be in the visible spectrum and may, for example, comprise fluorescent emissions. The reflected beam may comprise reflected emissions from an LED or a broad spectrum lamp, such as a mercury lamp (e.g. a high pressure mercury lamp) or a xenon arc lamp. The imaging device may further include a microscope (or optical train) which may or may not include an eyepiece.

A support structure. The system 150 may also include a support structure 190 configured to support and/or hold the housing 102 including the microfluidic circuit 120. In some embodiments, optional tilt module 166 may be configured to move support structure 190 to rotate microfluidic device 100 about one or more axes of rotation. Optional tilt module 166 may be configured to support and/or maintain microfluidic device 100 in a horizontal orientation (i.e., 0 ° with respect to the x-axis and y-axis), a vertical orientation (i.e., 90 ° with respect to the x-axis and/or y-axis), or any orientation therebetween. The orientation of the microfluidic device 100 (and microfluidic circuit 120) relative to the axis is referred to herein as the "tilt" of the microfluidic device 100 (and microfluidic circuit 120). For example, the support structure 190 can optionally be used to tilt the microfluidic device 100 (e.g., as controlled by optional tilt module 166) to 0.1 °, 0.2 °, 0.3 °, 0.4 °, 0.5 °, 0.6 °, 0.7 °, 0.8 °, 0.9 °, 1 °, 2 °, 3 °, 4 °, 5 °, 10 °, 15 °, 20 °, 25 °, 30 °, 35 °, 40 °, 45 °, 50 °, 55 °, 60 °, 65 °, 70 °, 75 °, 80 °, 90 °, or any angle therebetween, relative to the x-axis. When the microfluidic device is tilted at an angle greater than about 15 °, the tilting can be performed to produce a large amount of movement of the micro-objects from/to the flow region (e.g., microfluidic channel) into/from the isolation pens. In some embodiments, the support structure 190 can hold the microfluidic device 100 at a fixed angle of 0.1 °, 0.2 °, 0.3 °, 0.4 °, 0.5 °, 0.6 °, 0.7 °, 0.8 °, 0.9 °, 1 °, 2 °, 3 °, 4 °, 5 °, or 10 ° relative to the x-axis (horizontal) as long as the DEP is an effective force to move a micro-object out of an isolation fence into a microfluidic channel. Since the surface of the electrode activation substrate is substantially flat, DEP forces can be used even when the distal end of the isolation fence opposite its opening into the microfluidic channel is positioned lower in the vertical direction than the microfluidic channel.

In some embodiments where the microfluidic device is tilted or held at a fixed angle relative to horizontal, the microfluidic device 100 can be positioned in an orientation such that the inner surface of the base of the flow path 106 is positioned at an angle above or below the surface of the base of one or more isolation pens that open horizontally to the flow path. As used herein, the term "above" means that the flow path 106 is positioned higher than the one or more isolation pens on the vertical axis defined by gravity (i.e., objects in the isolation pens located above the flow path 106 will have a higher gravitational potential energy than objects in the flow region/channel) and, conversely, for positioning the flow path 106 below the one or more isolation pens. In some embodiments, the support structure 190 can be held at a fixed angle of less than about 5 °, about 4 °, about 3 °, or about 2 ° relative to the x-axis (horizontal), thereby placing the isolation pen at a lower potential energy relative to the flow path. In some other embodiments, when long-term culturing is performed within the microfluidic device (e.g., for more than about 2, 3, 4,5, 6, 7, or more days), the device can be supported on a culture carrier and can be tilted at a greater angle of about 10 °, 15 °, 20 °, 25 °, 30 °, or any angle therebetween, during the long-term culturing period to retain the biological micro-object within the isolation pen. At the end of the incubation period, the microfluidic device containing the incubated biological micro-objects can be returned to the support 190 within the system 150, where the angle of inclination is reduced to the value described above, thereby providing for the use of DEP to move the biological micro-objects out of the isolation pen. Other examples of using gravity caused by tilting are described in U.S. patent No. 9,744,533 (breinnlinger et al), which is incorporated herein by reference in its entirety.

And (6) nesting. Turning now to fig. 5A, the system 150 can include a structure (also referred to as a "nest") 500 configured to hold a microfluidic device 520 (which can be similar to the microfluidic devices 100, 200, or any other microfluidic device described herein). Nest 500 can include a receptacle 502 capable of interfacing with a microfluidic device 520 (e.g., photovoltaic powered device 100, 200, etc.) and providing an electrical connection from power source 192 to microfluidic device 520. Nest 500 may also include an integrated electrical signal generation subsystem 504. The electrical signal generation subsystem 504 may be configured to provide a bias voltage to the receptacle 502 such that the bias voltage is applied across a pair of electrodes in the microfluidic device 520 when the microfluidic device 520 is held by the receptacle 502. Thus, the electrical signal generation subsystem 504 may be part of the power supply 192. The ability to apply a bias voltage to the microfluidic device 520 does not mean that the bias voltage will always be applied when the microfluidic device 520 is held by the receptacle 502. Rather, in most cases, the bias voltage will be applied intermittently (e.g., only when needed) to facilitate the generation of electrokinetic forces (e.g., dielectrophoresis or electrowetting) in the microfluidic device 520.

As shown in fig. 5A, nest 500 may include a Printed Circuit Board Assembly (PCBA) 522. The electrical signal generation subsystem 504 may be mounted on and electrically integrated into the PCBA 522. The example support also includes a socket 502 mounted on the PCBA 522.

In some embodiments, the nest 500 may include an electrical signal generation subsystem 504 configured to measure the amplified voltage at the microfluidic device 520 and then adjust its own output voltage as needed so that the measured voltage at the microfluidic device 520 is a desired value. In some embodiments, the waveform amplification circuit may have a +6.5V to-6.5V power supply generated by a pair of DC-DC converters mounted on the PCBA 322, producing up to 13Vpp of signals at the microfluidic device 500.

In certain embodiments, nest 500 also includes a controller 508, such as a microprocessor for sensing and/or controlling electrical signal generating subsystem 504. Examples of suitable microprocessors include Arduino^TMMicroprocessors, e.g. Arduino Nano^TM. The controller 508 may be used to perform functions and analyses, or may communicate with an external master controller 154 (shown in FIG. 1A) to perform functions and analyses. In the embodiment shown in fig. 3A, the controller 308 communicates with the main controller 154 (of fig. 1A) through an interface (e.g., a plug or connector).

As shown in fig. 5A, the support structure 500 (e.g., nest) may further include a thermal control subsystem 506. Thermal control subsystem506 may be configured to regulate the temperature of a microfluidic device 520 held by the support structure 500. For example, the thermal control subsystem 506 may include a Peltier thermoelectric device (not shown) and a cooling unit (not shown). In the embodiment shown in fig. 5A, the support structure 500 includes an inlet 516 and an outlet 518 to receive cooling fluid from an external reservoir (not shown) of the cooling unit, introduce the cooling fluid into the fluid path 514 and through the cooling block, and return the cooling fluid to the external reservoir. In some embodiments, the peltier thermoelectric device, the cooling unit, and/or the fluid path 514 may be mounted on the housing 512 of the support structure 500. In some embodiments, the thermal control subsystem 506 is configured to adjust the temperature of the peltier thermoelectric generation device in order to achieve a target temperature for the microfluidic device 520. Temperature regulation of Peltier thermoelectric devices may be achieved, for example, by means such as Pololu ^TMThermoelectric power supplies (Pololu robotics and electronics). The thermal control subsystem 506 may include feedback circuitry, such as temperature values provided by analog circuitry. Alternatively, the feedback circuit may be provided by a digital circuit.

Nest 500 may include a serial port 524 that allows the microprocessor of controller 508 to communicate with external master controller 154 via an interface. Additionally, the microprocessor of the controller 508 may be in communication with the electrical signal generation subsystem 504 and the thermal control subsystem 506 (e.g., via a Plink tool (not shown)). Thus, the electrical signal generation subsystem 504 and the thermal control subsystem 506 may communicate with the external master controller 154 through a combination of the controller 508, the interface, and the serial port 524. In this manner, the main controller 154 may assist the electrical signal generation subsystem 504, among other things, by performing scaling calculations for output voltage adjustments. A Graphical User Interface (GUI) (not shown) provided via the display device 170 coupled to the external master controller 154 may be configured to plot the temperature and waveform data obtained from the thermal control subsystem 506 and the electrical signal generation subsystem 504, respectively. Alternatively or additionally, the GUI may allow for updating the controller 508, thermal control subsystem 506, and electrical signal generation subsystem 504.

An optical subsystem. Fig. 5B is a schematic diagram of an optical subsystem 550 having an optical device 510, the optical device 510 for imaging and manipulating micro-objects in a microfluidic device 520, the microfluidic device 520 may be any of the microfluidic devices described herein. The optical device 510 may be configured to perform imaging, analysis, and manipulation of one or more micro-objects within the housing of the microfluidic device 520.

The optical device 510 may also have a first light source 552, a second light source 554, and a third light source 556. The first light source 552 may transmit light to a structured light modulator 560, which structured light modulator 560 may include a Digital Mirror Device (DMD) or a micro shutter array system (MSA), either of which may be configured to receive light from the first light source 552 and selectively transmit a subset of the received light into the optical device 510. Alternatively, structured light modulator 560 may include an integrated device that produces its own light (and thus does not require light source 552), such as an organic light emitting diode display (OLED), a Liquid Crystal On Silicon (LCOS) integrated device, a ferroelectric liquid crystal on silicon integrated device (FLCOS), or a transmissive Liquid Crystal Display (LCD). The structured light modulator 560 may be, for example, a projector. Thus, the structured light modulator 560 is capable of emitting both structured light and unstructured light. In certain embodiments, the imaging module and/or the power module of the system may control the structured light modulator 560.

In an embodiment, when structured light modulator 560 includes a mirror, the modulator may have multiple mirrors. Each mirror of the plurality of mirrors may have a dimension of about 5 microns by 5 microns to about 10 microns by 10 microns or any value therebetween. Structured light modulator 560 may include an array of mirrors (or pixels) of 2000 x 1000, 2580 x 1600, 3000 x 2000, or any value in between. In some embodiments, only a portion of the illuminated area of the structured light modulator 560 is used. Structured light modulator 560 may transmit a selected subset of light to first dichroic beam splitter 558, which first dichroic beam splitter 558 may reflect the light to first barrel lens 562.

First tube lens 562 can have a large clear aperture, e.g., a diameter of greater than about 40mm to about 50mm or more, providing a large field of view. Accordingly, the first tube lens 5621 may have an aperture large enough to capture all (or substantially all) of the light beam emitted from the structured light modulator 560.

Alternatively or additionally, structured light 515 having a wavelength of about 400nm to about 710nm may provide fluorescence excitation illumination to the microfluidic device.

The second light source 554 may provide unstructured bright field illumination. The bright field illumination light 525 may have any suitable wavelength, and in some embodiments, may have a wavelength of about 400nm to about 760 nm. The second light source 554 may transmit light to the second dichroic beamsplitter 564 (which may also receive light 535 from the third light source 556), and the second light field illumination 525 may be transmitted therefrom to the first dichroic beamsplitter 558. Second bright field illumination 525 may then be transmitted from first beam splitter 558 to first tube lens 562.

The third light source 556 may transmit light to the reflector 566 via a matched relay lens (not shown). The third light illumination 535 can be reflected therefrom to the second dichroic beam splitter 5338, transmitted therefrom to the first beam splitter 5338, and extend forward to the first tube lens 5381. The third illumination light 535 may be a laser and may have any suitable wavelength. In some embodiments, the laser illumination 535 may have a wavelength of about 350nm to about 900 nm. The laser illumination 535 can be configured to heat portions of one or more isolation pens within the microfluidic device. The laser illumination 535 may be configured to heat a fluidic medium, a wall or a portion of a wall of an isolation pen, a metal target disposed in a microfluidic channel or an isolation pen of a microfluidic channel, or a light-reversible physical fence in a microfluidic device, and is described in more detail in U.S. application publication nos. 2017/0165667(Beaumont et al) and 2018/0298318(Kurz et al), the disclosures of each of which are incorporated herein by reference in their entirety. In other embodiments, the laser illumination 535 can be configured to initiate photo-lysis of surface-modified portions of modified surfaces of the microfluidic device or portions that provide adhesion functionality to micro-objects within isolation pens within the microfluidic device. More details of photocleavage using a laser can be found in international application publication No. WO2017/205830 (Lowe, jr. et al), the disclosure of which is incorporated herein by reference in its entirety.

The light from the first, second, and third light sources (552, 554, 5560) passes through the first tube lens 562 and is transmitted to the third dichroic beam splitter 568 and the color filter converter 572. The third dichroic beamsplitter 568 may reflect a portion of the light and transmit the light to the objective 570 through one or more color filters in a color filter converter 572, which may be an objective changer with a plurality of different objectives that may be switched as desired. Some of the light (515, 525, and/or 535) may pass through the third dichroic beamsplitter 568 and be terminated or absorbed by a beam block (not shown). Light reflected from third dichroic beamsplitter 568 passes through objective lens 570 to illuminate sample plane 574, which can be part of microfluidic device 520 (e.g., an isolation pen as described herein).

As shown in fig. 5A, nest 500 may be integrated with optical device 510 and may be part of device 510. The nest 500 can provide electrical connections to the housing and can also be configured to provide fluid communication with the housing. A user can load the microfluidic device 520 into the nest 500. In some other embodiments, nest 500 may be a separate component from optical device 510.

Light may be reflected and/or emitted from the sample plane 574 to pass through the objective lens 570, through the color filter converter 572, and back through the third dichroic beamsplitter 568 to the second tube lens 576. The light may pass through the second tube lens 576 (or the imaging tube lens 576) and reflect from the mirror 578 to the imaging sensor 580. Stray light baffles (not shown) may be placed between the first tube lens 562 and the third dichroic beam splitter 568, between the third dichroic beam splitter 568 and the second tube lens 576, and between the second tube lens 576 and the imaging sensor 580.

An objective lens. The optical device may include an objective 570 specifically designed and configured for viewing and manipulating micro-objects in the microfluidic device 520. For example, a conventional microscope objective is designed to view micro-objects on a slide or by 5mm aqueous fluid (aqueous fluid), while the micro-objects in the microfluidic device 520 have a depth of 20, 30, 40, 50, 60, 70, 80 microns or any value in between when inside the plurality of isolated pens in the observation plane 574. In some embodiments, a transparent cover 520a (e.g., a glass or ITO cover having a thickness of about 750 microns) can be placed on top of a plurality of isolation fences disposed over microfluidic substrate 520 c. Therefore, images of micro-objects obtained by using conventional microscope objectives may have large aberrations (e.g., spherical and chromatic aberrations), which may reduce the quality of the images. The objective lens 570 of the optical device 510 may be configured to correct spherical and chromatic aberrations in the optical device 1350. Objective 570 may have one or more available magnifications, e.g., 4X, 10X, 20X.

An illumination mode. In some embodiments, the structured light modulator 560 can be configured to modulate the light beam received from the first light source 552 and transmit the plurality of illumination beams 515 as a structured light beam into the housing of the microfluidic device, e.g., an area containing an insulated pen. The structured light beam may comprise a plurality of illuminating light beams. The plurality of illumination beams may be selectively activated to produce a plurality of illumination patterns. In some embodiments, the structured light modulator 560 may be configured to produce an illumination pattern that may be moved and adjusted similar to that described with respect to fig. 4A-4B. The optical device 560 can also include a control unit (not shown) configured to adjust the illumination pattern to selectively activate one or more of the plurality of DEP electrodes of the substrate 520c and generate DEP forces to move one or more micro-objects in the plurality of isolated pens within the microfluidic device 520. For example, multiple illumination patterns may be adjusted in a controlled manner over time to manipulate micro-objects in the microfluidic device 520. Each of the plurality of illumination patterns may be offset to shift the position of the generated DEP force and move the structured light from one position to another in order to move micro-objects within the housing of the microfluidic device 520.

In some embodiments, the optical device 510 can be configured such that each of a plurality of isolated pens in the sample plane 574 within the field of view is focused at the image sensor 580 and the structured light modulator 560 simultaneously. In some embodiments, the structured light modulator 560 may be disposed at a conjugate plane of the image sensor 580. In various embodiments, the optical device 510 may have a confocal configuration or confocal characteristics. The optical device 510 can also be configured such that only each of a plurality of isolated pens in the sample plane 574 within each interior region and/or field of view of the flow region is imaged onto the image sensor 580 to reduce overall noise, thereby improving the contrast and resolution of the image.

In some embodiments, first tube lens 562 may be configured to generate a collimated beam and transmit the collimated beam to objective lens 570. Objective 570 may receive and focus the collimated beam from first tube lens 562 in each interior region of the flow region and in each of a plurality of isolated pens in sample plane 574 within the field of view of image sensor 580 or optical device 510. In some embodiments, first tube lens 562 may be configured to generate and transmit a plurality of collimated light beams to objective lens 570. Objective 570 may receive the plurality of collimated light beams from first tube lens 562 and converge the plurality of collimated light beams in each of a plurality of isolated pens in sample plane 574 within the field of view of image sensor 580 or optical device 510.

In some embodiments, the optical device 510 can be configured to illuminate at least a portion of the insulated pen with a plurality of illumination points. Objective 570 may receive the plurality of collimated light beams from first tube lens 562 and project a plurality of illumination points, which may form an illumination pattern, into each of a plurality of isolated pens in sample plane 574 within the field of view. For example, each of the plurality of irradiated spots may have a size of about 5 microns X5 microns, 10 microns X10 microns, 10 microns X30 microns, 30 microns X60 microns, 40 microns X40 microns, 40 microns X60 microns, 60 microns X120 microns, 80 microns X100 microns, 100 microns X140 microns, and any value therebetween. The illumination spots may have a circular, square or rectangular shape, respectively. Alternatively, the illumination points may be grouped within a plurality of illumination points (e.g., illumination patterns) to form larger polygonal shapes, such as rectangles, squares, or wedges. The illumination pattern may encompass (e.g., surround) a space that may be square, rectangular, or polygonal that is not illuminated. For example, each of the plurality of irradiated spots may have an area of about 150 to about 3000, about 4000 to about 10000, or 5000 to about 15000 square microns. The illumination pattern may have an area of about 1000 to about 8000, about 4000 to about 10000, 7000 to about 20000, 8000 to about 22000, 10000 to about 25000 square microns, and any value therebetween.

The optical system 510 can be used to determine how to relocate micro-objects into and out of the isolation pens of the microfluidic device, as well as to count the number of micro-objects present in the microfluidic circuit of the device. More details of repositioning and counting micro-objects are found in the following documents: U.S. application publication No. 2016/0160259 (Du), U.S. patent No. 9,996,920 (Du et al), and international application publication No. WO2017/102748 (Kim et al). The optical system 510 may also be used in an assay method to determine the concentration of a reagent/assay product, and further details are found in: U.S. patent No. 8,921,055 (Chapman), U.S. patent No. 10,010,882 (White et al), U.S. patent No. 9,889,445 (Chapman et al), international application publication No. WO2017/181135 (Lionberger et al), international application serial No. PCT/US2018/055918 (Lionberger et al). Further details of the features of optical devices suitable for use within systems for viewing and manipulating micro-objects within microfluidic devices as described herein may be found in WO2018/102747(Lundquist et al), the disclosure of which is incorporated herein by reference in its entirety.

A cell. The cells that can be used in the systems and methods of the present disclosure can be any type of plant protoplast. For example, protoplasts can be from any type of plant used in agriculture. Non-limiting examples of agricultural plants include: large area crops, such as wheat, corn, soybean or cotton plants; high value crops, such as tobacco, tomato, lettuce, pepper or squash plants; brassica plants, such as broccoli, mustard, brussels sprouts, cabbage, cauliflower, kale, kohlrabi, oilseed rape, kohlrabi, turnip or arabidopsis thaliana plants; ornamental plants, for example, rose, petunia, poppy, lilac, lavender, silvergrass or cactus plants; fruit trees, shrubs or vines, e.g., grapes, apples, oranges, strawberries, blackberries, blueberries, raspberries, plums, luffa, apricots, and the like; or turf or forage plants, e.g., grass or alfalfa plants. Methods for obtaining protoplasts are known in the art and have been described, for example, in the following references: giles, Kenneth, editors, Plant Protoplasts, International Review of Cytology (Plant Protoplasts: International reviews), Vol 16, Academic Press 1983; yoo et al (2007), Nature Protocols, Vol.2 (7), 1565-72; and Danon (2014), Bio-Protocol, Vol.4 (12), e 1149.

In some embodiments, the cells may be from a cell population that is actively growing in culture or obtained from a fresh tissue sample (e.g., by isolating a solid tissue sample, such as a plant leaf, stem, root, flower, etc.). Alternatively, one or more biological cells may be from a culture of other samples that have been previously frozen.

Depending on the particular objective of the experiment, only one or more cells may be introduced into the growth chamber (e.g., isolation pen) of the microfluidic device for culture and/or cloning. When only one cell is introduced into the growth chamber of the system and incubated according to the methods described herein, the resulting expanded population is a clonal colony of cells that were initially introduced into the growth chamber.

A method. A method for culturing and assaying at least one cell, in particular a plant protoplast, in a system comprising a microfluidic device having at least one growth chamber and a flow region is provided. Culturing cells in a microfluidic growth chamber having a volume on the nanoliter scale can facilitate the culturing of cells that would otherwise be uncultured. For example, an effective concentration of a single cell in a chamber of 1 nanoliter volume is 1x10⁶cells/mL. Due to the small volume of the chamber, proteins and other molecules released into the culture can rapidly regulate the medium in the chamber, thereby ensuring that the cells receive the signals needed to support cell viability. In addition, having flow areas Culturing cells in a growth chamber of a microfluidic device may allow for the specific introduction of nutrients, growth factors, or other cell signaling species over a selected period of time to achieve control over cell growth, viability, or transplantable parameters. The precise control of possible cell placement/removal and nutrient/signal/environmental stimuli by the methods described herein makes it difficult or impossible to achieve by large-scale culture or other microfluidic culture methods.

At least one biological cell (e.g., a plant protoplast) can be introduced into a growth chamber having at least one conditioned surface, where the conditioned surface supports cell growth, viability, portability, or any combination thereof, as described above. In some embodiments, the conditioned surface supports cell portability within the microfluidic device. In some embodiments, portability includes preventing non-specific adhesion of cells to the microfluidic device. The at least one conditioned surface may be any conditioned surface described herein. The conditioned surface can be covalently attached to a microfluidic device. In some embodiments, the conditioned surface may include a linking group covalently attached to the surface, and the linking group may also be attached to a moiety configured to support cell growth, viability, portability, or any combination thereof, of one or more biological cells in the microfluidic device. In some embodiments, a microfluidic device having a conditioned surface may be provided prior to introduction of one or more biological cells. As described herein, introduction of biological cells can be accomplished using a number of different motives, some of which can allow precise control over the placement of particular biological cells in particular locations on the microfluidic device, such as in pre-selected growth chambers.

After placement, the at least one biological cell is then incubated for at least a sufficient period of time to expand the at least one biological cell to produce a biological cell colony. When biological cells (e.g., plant protoplasts) are introduced into separate growth chambers, the resulting expanded colonies can be accurately identified for further use as an isolatable biological cell population. When only one biological cell is introduced into the growth chamber and allowed to expand, the resulting colony is a clonal population of biological cells. Any suitable cell may be used in the method, including but not limited to the cells described above.

The microfluidic device may be any of the microfluidic devices 100, 300, 400, 500A-E, or 600 described herein, and the microfluidic device may be part of a system having any of the components described herein. The at least one growth chamber may comprise a plurality of growth chambers, and any suitable number of growth chambers discussed herein may be used.

Introducing at least one biological cell. In some embodiments, introducing at least one biological cell (e.g., a plant protoplast) into at least one growth chamber can include moving the at least one biological cell using a Dielectrophoretic (DEP) force of sufficient strength. DEP forces can be generated using electronic tweezers (e.g., optoelectronic tweezers (OET)). In some other embodiments, introducing one or more biological cells into at least one growth chamber can include using fluid flow and/or gravity (e.g., by tilting the microfluidic device to drop the cells into the growth chamber below the cells).

In some embodiments, at least one biological cell (e.g., a plant protoplast) is introduced into a flow region (e.g., a flow channel) of a microfluidic device through the inlet port 124. The flow of the medium in the flow channel can carry the cells to a location proximate to the opening of the growth chamber. After being in an open position proximate to the growth chamber, the biological cells can then be moved into the growth chamber using any of the motive forces described herein, including dielectrophoresis or gravity. Dielectrophoretic forces may comprise electrically or optically actuated forces, and DEP forces may also be provided by optoelectronic tweezers (OET). The at least one biological cell may be moved through the flow channel to a proximal opening of a communication region of the at least one growth chamber, wherein the communication region opens directly into and is in fluid communication with the flow channel/region. The communicating region of the at least one growth chamber is also in fluid communication with the isolated region of the at least one growth chamber. The at least one biological cell may also move through the communication zone and into the isolation zone of the at least one growth chamber. The isolation region of the at least one growth chamber may haveA size sufficient to support cell expansion. However, in general, the size of the growth chamber limits this expansion to no more than about 1x10 in culture ²50, 25, 15 or even at least 10 cells. In some embodiments, the isolation region can have a volume sufficient to support expansion of cells in culture to no more than about 1 × 10²50, 25, 15 or 10 cells in size. It has surprisingly been found that the volume does not exceed 1.5X 10⁶Cubic micron or 1.0X 10⁶Incubation and/or expansion of protoplasts of up to about 20 cells or more can be successfully performed in isolated regions of cubic micrometers. Cell diameters can vary widely depending on the type of protoplast. Thus, it has a size of about 5X 10⁵A cubic micron volume growth chamber may allow only a few protoplasts with large diameters (e.g., about 30 microns to about 50 microns in diameter) to expand while the same small growth chamber (about 5X 10 in volume)⁵Cubic microns) may allow for greater amplification of protoplasts having smaller diameters (e.g., about 10 microns to about 30 microns in diameter).

The method may further comprise introducing a first fluidic medium into a microfluidic channel of a flow region of the microfluidic device. In some embodiments, the introduction of the first fluid medium is performed prior to the introduction of the at least one plant protoplast. When the first fluidic medium is introduced prior to the introduction of the at least one plant protoplast, the flow rate can be selected such that the first fluidic medium flows from the flow channel of the microfluidic device into the growth chamber at any suitable rate. Alternatively, if the microfluidic device has been filled with a medium containing an excess of one or more conditioning agents, the first fluidic medium flows into the microfluidic channel at a rate such that the first fluidic medium displaces any remaining medium containing an excess of conditioning agents in the flow region.

When introducing the flow of the first fluid medium after introducing the at least one plant protoplast into the growth chamber, the flow rate of the first fluid medium can be selected to not sweep the isolation area, which will not cause the at least one plant protoplast to leave the isolation area. The fluid medium surrounding the at least one plant protoplast in the isolated region of the at least one growth chamber is a second fluid medium, which can be the same or different from the first fluid medium. In some embodiments, the second fluid medium may be the same as the first fluid medium, but during the incubation step, the cellular waste and depleted medium components may cause the second fluid medium to be different from the first fluid medium.

The cells were incubated. In the methods described herein, at least one plant protoplast is incubated for at least a period of time sufficient for the cells to expand to produce a colony of biological cells. The time period may be selected to be about 1 day to about 14 days. In other embodiments, the incubation period may extend over 14 days and may last for any desired period of time. Since the cells in the isolated region of the growth chamber are provided with nutrients and waste products are removed by the perfusion of the fluid medium, the cells can grow indefinitely. Since the isolation zone is filled with an expanded cell population, any additional expansion will result in the expanded plant protoplasts residing in the communicating region of the growth chamber, which is the swept region of the growth chamber. The medium to be perfused may be any medium suitable for culturing or maintaining plant protoplasts. Suitable protoplast media are known in the art. See, e.g., Giles, Kenneth, editors, Plant Protoplasts, International Review of biology (Plant Protoplasts: International reviews), volume 16, Academic Press 1983; yoo et al (2007), Nature Protocols, Vol.2 (7), 1565-72; and Danon (2014), Bio-Protocol, Vol.4 (12), e 1149.

The perfused medium may sweep the amplified protoplasts across a contiguous region of the growth chamber and then out of the microfluidic device. Thus, the number of protoplasts present in the isolated region of the growth chamber can be stabilized at a maximum number that depends on the size of the protoplasts and the size of the isolated region of the growth chamber. The ability to stabilize the maximum number of cells in an isolated cell population provides an advantage over other currently available cell culture methods in that tedious segregation of cell populations can be eliminated.

In some embodiments, the incubation can be performed for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days or longer. The incubation period can range from about 1 day to about 6 days, from about 1 day to about 5 days, from about 1 day to about 4 days, from about 1 day to about 3 days, or from about 1 day to about 2 days. In other embodiments, the incubation may be performed for less than about 5 days, less than about 4 days, less than about 3 days, or less than about 2 days. In some embodiments, the incubation can be performed for less than about 3 days or less than about 2 days. In other embodiments, the incubation can be performed for about 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, or about 23 hours.

During the culturing step, images of at least one growth chamber and any cells contained therein can be monitored at one or more time points throughout the culturing step. The image may be stored in a memory of a processing component of the system.

Cells were perfused. During the incubation step, the second fluid medium present within the isolated region of the growth chamber may be depleted of nutrients, growth factors, or other growth stimulants. The second fluid medium may accumulate cellular waste. In addition, as at least one cell (e.g., a plant protoplast) continues to grow during the incubation, it may be desirable to change the nutrient, growth factor, or other growth stimulator to a nutrient, growth factor, or other growth stimulator that is different from the first or second medium at the beginning of the incubation. As described herein, culturing in the growth chamber of a microfluidic device can provide the specific and selective ability to introduce and alter a chemical gradient sensed by at least one plant protoplast, which can more closely approximate in vivo conditions. Alternatively, changing the chemical gradient sensed by at least one biological cell to a purposeful set of non-optimal conditions may allow the cell to expand under conditions designed to explore a disease or therapeutic pathway. Thus, the method may comprise perfusing the first fluid medium during the incubation step, wherein the first fluid medium is introduced via the at least one inlet 124 of the microfluidic device, and wherein the first fluid medium, optionally comprising components from the second fluid medium, is output via the at least one outlet of the microfluidic device.

Exchange of components of the first fluid medium to provide fresh nutrients, soluble growth factors, etc., and/or exchange of waste components of the medium around the cells within the isolation zone occurs substantially under diffusion conditions at the interface of the swept and unswept areas of the growth chamber. It has surprisingly been found that effective exchange occurs in the substantial absence of flow. Thus, it was surprisingly found that successful incubation did not require continuous perfusion. Thus, the perfusion may be discontinuous. In some embodiments, the perfusion is periodic, and in some embodiments, the perfusion is aperiodic. The interruption between perfusion cycles may be of sufficient duration to allow diffusion of the component of the second fluid medium in the isolation zone into the first fluid medium in the flow channel/zone and/or diffusion of the component of the first fluid medium into the second fluid medium without substantially all of the first medium flowing into the isolation zone.

In another embodiment, a low perfusion rate may also be employed to obtain efficient exchange of components of the fluid medium inside and outside the unswept area of the growth chamber.

Thus, one method of perfusing at least one biological cell in at least one growth chamber of a microfluidic device is shown in fig. 6, the method comprising a perfusion step 6002 in which a first fluidic medium is passed through a flow region of the microfluidic device at a first perfusion rate R1 into the flow region in fluid communication with the growth chamber for a first perfusion time D1. As described herein, R1 may be selected as the unswept flow rate. The method 600 further comprises a step 6004, wherein the flow of fluidic medium is stopped for a first perfusion stop time S1. Steps 6002 and 6004 are repeated a number of times W, where W may be an integer selected from 1 to about 1000, whereupon the perfusion process 700 is complete. In some embodiments, W may be an integer from 2 to about 1000.

Another method 700 of perfusing at least one biological cell in at least one growth chamber of a microfluidic device is shown in fig. 7, the method comprising a first perfusion cycle comprising step 7002 in which a fluidic medium is flowed through a flow region of the microfluidic device at a first perfusion rate R1 into the flow region fluidly connected to the growth chamber for a first perfusion time D1. As described herein, R1 may be selected as the unswept flow rate. The first perfusion cycle includes step 7004 of stopping the flow of the fluidic medium for a first perfusion stop time S1. The first perfusion cycle may be repeated W times, where W is an integer selected from 1 to about 1000. After completing the W-th iteration of the first perfusion cycle, method 700 further comprises a second perfusion cycle comprising the step 7006 of flowing the first fluid medium at a second perfusion rate R2 for a second perfusion time D2, wherein R2 is selected as the unswept flow rate. The second perfusion cycle of method 700 further includes step 7008 of stopping the flow of the fluidic medium for a second perfusion stop time S2. Thereafter, the method returns to steps 7002 and 7004 of the first perfusion cycle and repeats the combined two-cycle perfusion process V times, where V is an integer from 1 to about 5000. The combination of W and V may be selected to meet the desired end of the incubation period.

In various embodiments of methods 600 or 700, perfusion rate R1 may be any unswept flow rate of the fluidic medium as described above for the flow controller configuration. In some embodiments, R1 may be about 0.009, 0.010, 0.020, 0.030, 0.040, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 1.00, 1.10, 1.20, 1.30, 1.40, 1.50, 1.60, 1.70, 1.80, 1.90, 2.00, 2.10, 2.20, 2.40, 2.50, 2.60, 2.70, 2.80, 2.90, or 3.00 microliters/second.

In various embodiments of method 700, the second perfusion rate R2 may be any unswept flow rate of the fluidic medium as described above for the flow controller configuration. In some embodiments, R2 may be 0.009, 0.010, 0.020, 0.030, 0.040, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 1.00, 1.10, 1.20, 1.30, 1.40, 1.50, 1.60, 1.70, 1.80, 1.90, 2.00, 2.10, 2.20, 2.40, 2.50, 2.60, 2.70, 2.80, 2.90, or 3.00 microliters/second. The flow rates R1 and/or R2 may be selected in any combination. Generally, the perfusion rate R2 may be greater than the perfusion rate R1, and may be about 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or greater than R1. In some embodiments, R2 is at least ten times faster than R1. In other embodiments, R2 is at least twenty times faster than R1. In yet another embodiment, R2 is at least 100 times the rate of R1.

In various embodiments of methods 600 or 700, the first priming time Dl may be any suitable priming duration as described above for the flow controller configuration. In various embodiments, D1 may be about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, or 180 seconds. In other embodiments, D1 may be a time range, for example, about 10 seconds to about 40 seconds, as described above. In some embodiments, D1 may be about 30 seconds to about 75 seconds. In other embodiments, D1 may be about 100 seconds. In other embodiments, D1 may range from about 60 seconds to about 150 seconds. In other embodiments, D1 may be about 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 80 minutes, 90 minutes, 110 minutes, 120 minutes, 140 minutes, 160 minutes, 180 minutes, 200 minutes, 220 minutes, 240 minutes, 250 minutes, 260 minutes, 270 minutes, 290 minutes, or 300 minutes. In some embodiments, D1 is from about 40 minutes to about 180 minutes.

In various embodiments of methods 600 or 700, second perfusion time D2 may be any suitable perfusion duration as described above for the flow controller configuration. In various embodiments, D2 may be about 5 seconds, 10 seconds, 15 seconds, 20 seconds, 25 seconds, 30 seconds, 35 seconds, 40 seconds, 45 seconds, 50 seconds, 55 seconds, 60 seconds, 65 seconds, 70 seconds, 80 seconds, 90 seconds, or about 100 seconds. In other embodiments, D2 may be a time range, for example, about 5 seconds to about 20 seconds, as described above. In other embodiments, D2 may be about 30 seconds to about 70 seconds. In other embodiments, D2 may be about 60 seconds.

In various embodiments of methods 600 or 700, the first perfusion time D1 may be the same as or different from the second perfusion time D2. D1 and D2 may be selected in any combination. In some embodiments, the duration of the priming D1 and/or D2 may be selected to be shorter than the stopping time periods S1 and/or S2.

In various embodiments of the methods 600 or 700, the first perfusion stop time S1 may be selected as any suitable time period as described above for the time interval between perfusion time periods configured by the flow controller. In some embodiments, S1 may be about 0 minutes, 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 60 minutes, about 65 minutes, about 80 minutes, about 90 minutes, about 100 minutes, about 120 minutes, about 150 minutes, about 180 minutes, about 210 minutes, about 240 minutes, about 270 minutes, or about 300 minutes. In various embodiments, S1 may be any suitable time range, such as about 20 to 60 minutes, as described above for the flow controller configuration interval between infusions. In some embodiments, S1 may be from about 10 minutes to about 30 minutes. In other embodiments, S1 may be about 15 minutes. In other embodiments, S1 may be about 0 seconds, 5 seconds, 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, 60 seconds, 70 seconds, 80 seconds, or about 90 seconds. In some embodiments, S1 is about 0 seconds.

In various embodiments of methods 600 or 700, the second perfusion stop time S2 may be selected as any suitable time period as described above for the time interval between perfusion time periods configured by the flow controller. In some embodiments, S2 may be about 0 minutes, 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 50 minutes, about 60, about 90 minutes, about 120 minutes, about 180 minutes, about 240 minutes, about 270 minutes, or about 300 minutes. In various embodiments, S2 may be any suitable time range as described above for the flow controller configuration interval between infusions, e.g., about 15 to 45 minutes. In some embodiments, S2 may be from about 10 minutes to about 30 minutes. In other embodiments, S2 may be about 8 minutes or 9 minutes. In other embodiments, S2 is about 0 minutes.

In various embodiments of methods 600 or 700, the first perfusion stop time S1 and the second perfusion stop time S2 may be independently selected to be any suitable values. S1 may be the same as or different from S2.

In various embodiments of method 700, the number of W repetitions may be selected to be the same as or different from the number of V repetitions.

In various embodiments of the method 600 or 700, W may be about 1, about 4, about 5, about 6, about 8, about 10, about 12, about 15, about 18, about 20, about 24, about 30, about 36, about 40, about 45, or about 50. In some embodiments, W may be selected to be about 1 to about 20. In some embodiments, W may be 1.

In various embodiments of the method 700, V may be about 5, about 10, about 20, about 25, about 30, about 35, about 40, about 50, about 60, about 80, about 100, about 120, about 240, about 300, about 350, about 400, about 450, about 500, about 600, about 750, about 900, or about 1000. In some embodiments, V may be selected to be from about 10 to about 120. V may be from about 5 to about 24. In some embodiments, V may be from about 30 to about 50 or may be from about 400 to about 500.

In various embodiments of method 700, the number of W repetitions may be selected to be the same as or different from the number of V repetitions.

In various embodiments of methods 600 or 700, the total time for the first step of priming (represented by step 7002/7004 or 8002/8004) is about 1h to about 10h, and W is an integer 1. In various embodiments, the total time for the first step of priming is from about 9 minutes to about 15 minutes.

In various embodiments of the method 700, the total time for the second step of the perfusion cycle (represented by step 8006/8008) is from about 1 minute to about 15 minutes or from about 1 minute to about 20 minutes.

In any of methods 600 or 700, the perfusion method can be performed continuously throughout the incubation period of the biological cells, e.g., for about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10 days, or longer.

In another non-limiting embodiment of the method 700 of fig. 7, the controller may be configured to perfuse the fluidic medium in a flow region with a longer perfusion cycle D1 during the perfusion step 7002. The controller may perfuse the fluid medium at the first rate for a period of time of about 45 minutes, about 60 minutes, about 75 minutes, about 90 minutes, about 105 minutes, about 120 minutes, about 2.25 hours, about 2.5 hours, about 2.45 hours, about 3.0 hours, about 3.25 hours, about 3.5 hours, about 3.75 hours, about 4.0 hours, about 4.25 hours, about 4.5 hours, about 4.75 hours, about 5 hours, or about 6 hours. At the end of the first perfusion period D1, the flow of fluidic medium may be stopped for a stop period S1, which stop period S1 may be about 0 seconds, 15 seconds, 30 seconds, about 45 seconds, about 1 minute, about 1.25 minutes, about 1.5 minutes, about 2.0 minutes, about 3.0 minutes, about 4 minutes, about 5 minutes, or about 6 minutes. In some embodiments, the first flow rate R1 may be selected to be about 0.009, 0.01, 0.02, 0.03, 0.05, 0.1, 0.2, 0.3, 0.4, or about 0.5 microliters/second. The flow of fluidic medium may be selected to stop for a perfusion stop period of less than about 1 minute S1, or S1 may be 0 seconds. Alternatively, S1 may be about 30 seconds, about 1.5 minutes, about 2.0 minutes, about 2.5 minutes, or about 3 minutes. A second perfusion cycle D2 may follow using a different perfusion rate. In some embodiments, the second perfusion rate may be higher than the first perfusion rate. In some embodiments, the second perfusion rate R2 may be selected from about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.7, 1.9, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0, 6.0, 7.0, 8.0, or about 9.0 microliters/second. The second perfusion cycle D2 may be about 1 second, about 2 seconds, about 3 seconds, about 4 seconds, about 5 seconds, about 6 seconds, about 10 seconds, about 15 seconds, about 30 seconds, about 45 seconds, about 60 seconds, about 65 seconds, about 75 seconds, about 80 seconds, or about 90 seconds. Perfusion may then be stopped for a second perfusion stop period S2, which may be about 0 seconds, 10 seconds, about 20 seconds, about 30 seconds, about 40 seconds, about 50 seconds, about 60 seconds, about 1.5 minutes, about 1.75 minutes, about 2.0 minutes, about 2.5 minutes, about 2.75 minutes, about 3.0 minutes, or about 4.0 minutes, for the second perfusion stop period S2. In some embodiments, D1 may be about 2 hours, about 3 hours, or about 4 hours. In various embodiments, D1 may be about 4 hours. In various embodiments, S1 may be 0 seconds or less than about one minute. The second perfusion cycle D2 may be about 1 second to about 6 seconds. In some embodiments, the second perfusion stop period S2 may be about 40 seconds to about 1.5 minutes.

Accordingly, there is provided a method for perfusing at least one biological cell in at least one growth chamber of a microfluidic device, the method comprising the step of perfusing the at least one biological cell (e.g., a plant protoplast) using a first perfusion step comprising the steps of: flowing a first fluidic medium through a flow region of the microfluidic device at a first priming rate R1 for a first priming time D1, wherein the flow region is in fluid communication with the growth chamber, wherein R1 is selected as the unswept flow rate; stopping the flow of the first fluid medium for a perfusion stop time S1; and repeating the first perfusion step W times, wherein W is an integer selected from 1 to 1000. The method may further comprise the step of perfusing the at least one biological cell with a second perfusion step comprising the steps of: flowing the first fluid medium at a second perfusion rate R2 for a second perfusion time D2, wherein R2 is selected as the unswept flow rate; stopping the flow of the first fluidic medium for a second perfusion stop time S2; and repeating the first perfusion step and subsequently the second perfusion step V times, wherein V is an integer from 1 to 1000.

The second perfusion rate R2 may be greater than the first perfusion rate R1. The first perfusion time D1 may be the same as or different from the second perfusion time D2. The first perfusion stop time S1 may be the same as or different from the second perfusion stop time S2. When performing the second perfusion step, the number of W repetitions may be the same as or different from the number of V repetitions. R2 may be at least ten times faster than R1. Alternatively, R2 may be at least twenty times faster than R1. R2 may be at least 100 times faster than R1. D1 may be about 30 seconds to about 75 seconds. In other embodiments, D1 may be from about 40 minutes to about 180 minutes or from about 180 minutes to about 300 minutes. In some other embodiments, D1 may be about 60 seconds to about 150 seconds. S1 may be from about 10 minutes to about 30 minutes. In other embodiments, S1 may be from about 5 minutes to about 10 minutes. In other embodiments, S1 may be zero. In some embodiments, D1 may be about 40 minutes to about 180 minutes, and S1 may be zero. In other embodiments, D1 may be from about 60 seconds to about 150 seconds, and S1 may be from about 5 minutes to about 10 minutes. In other embodiments, D1 may be about 180 minutes to about 300 minutes, and S1 may be zero. The total time for the first perfusion step may be about 1 hour to about 10 hours. In other embodiments, the total time for the first perfusion step may be about 2 hours to about 4 hours. In some embodiments, W may be an integer greater than 2. In some embodiments, W may be from about 1 to about 20. In some embodiments, D2 may be about 10 seconds to about 25 seconds. In other embodiments, D2 may be about 10 seconds to about 90 seconds. In some embodiments, S2 may be from about 10 minutes to about 30 minutes. In other embodiments, S2 may be about 15 minutes. In some embodiments, V may be from about 10 to about 120. In some embodiments, V may be from about 30 to about 50 or may be from about 400 to about 500. In some embodiments, D2 may be from about 1 second to about 6 seconds. S2 may be 0 seconds. In some embodiments, D2 may be about 10 seconds to about 90 seconds, and S2 may be about 40 seconds to about 1.5 minutes. In some embodiments, the total time for one repetition of the second perfusion step may be about 1 minute to about 15 minutes.

A conditioning medium. To provide a medium (e.g., a first medium or a second medium) that maintains and enhances the growth and/or viability of at least one plant protoplast, the first fluid medium can comprise a liquid and a gaseous component (e.g., the gaseous component can be dissolved in the liquid component). In addition, the fluid medium may include other components, such as biomolecules, vitamins, and minerals dissolved in the liquid component. Any suitable component may be used in the fluid medium, as known to the skilled person. Some non-limiting examples are discussed above, but many other media compositions may be used without departing from the methods described herein. In some embodiments, the fluid medium may comprise a chemically-defined medium (at least prior to contacting the cells or the fluid containing the cells), and may also be a protein-free or peptide-free chemically-defined medium.

The first fluidic medium may be prepared by saturating the initial fluidic medium with dissolved gaseous molecules prior to introducing the first fluidic medium into the microfluidic device. In addition, the initial fluidic medium may be saturated with dissolved gaseous molecules until the point in time at which the first fluidic medium is introduced into the microfluidic device. Saturating the initial fluidic medium may include contacting the microfluidic device with a gaseous environment capable of saturating the initial fluidic medium with dissolved gaseous molecules. Gaseous molecules that may saturate the initial fluid medium include, but are not limited to, oxygen, carbon dioxide, and nitrogen.

The first fluid medium may further comprise adjusting the pH of the first fluid medium. Adjusting the pH of the first fluid medium may occur, for example, before and/or during introduction of the dissolved gaseous molecules. This adjustment can be achieved by adding a buffer substance. One non-limiting example of a suitable buffer substance is HEPES. Other buffering substances may be present in the medium, and may or may not depend on the presence of carbon dioxide (e.g., a carbonation buffering system), and may be selected by the skilled artisan. Salts, proteins, carbohydrates, lipids, vitamins and other small molecules required for cell growth may also form part of the first fluid medium composition.

In some embodiments, saturating the first fluid medium with the gaseous component may be performed in the reservoir prior to introduction via the inlet port. In other embodiments, the saturation of the first fluid medium by the gaseous component may be performed in a gas-permeable connecting conduit between the reservoir and the inlet. In other embodiments, saturating the first fluidic medium with the gaseous component can be performed via a gas permeable portion of a lid of the microfluidic device. In some embodiments, gaseous saturation of the fluid medium further comprises maintaining humidity in the gas exchange environment such that the fluid medium within the microfluidic device does not change osmolality during the incubation period.

The components of the first fluid medium may further comprise at least one secreted component from the feeder cell culture. Secreted feeder cell components may include growth factors, hormones, cytokines, small molecules, proteoglycans, and the like. The introduction of the at least one secretory component from the feeder cell culture may be carried out in the same reservoir in which the saturation of the first fluid medium with the gaseous component is carried out, or the introduction of the at least one secretory component from the feeder cell culture into the first fluid medium may be carried out prior to the saturation step.

In some other embodiments, the components of the first medium may further include an additive designed to provide an altered fluid medium to test the response of the cells to the additive. Such additives may, for example, increase or decrease cell viability or growth.

In some embodiments, the method may comprise detecting the pH of the first fluid medium when the first fluid medium is introduced via the at least one inlet. The pH measurement may be performed at a location proximate to the inlet. In some embodiments, the method may include detecting a pH of the first fluid medium while outputting the first fluid medium via the outlet. The pH detection may be performed at a location proximate to the outlet. One or both of the detectors for detecting pH may be an optical sensor. In some embodiments, the detector can provide an alert if the pH deviates from an acceptable range. In some other embodiments, the composition of the first fluid medium may be changed when the pH measured by the detector deviates from an acceptable range.

During the incubation step, images of the at least one growth chamber and any cells contained therein may be monitored.

And (4) screening plant protoplasts. Plant protoplasts can be screened for disease resistance by contacting the protoplasts with a pathogen or fragment thereof and monitoring the plant protoplasts to determine whether they remain viable. Exemplary screening is described in example 3 below, as well as in the examples and claims listed below. Plant immunization is generally described, for example, in the following documents: boutrot and Zipfel (2017), annu. rev. phytopathohol.55: 257-86; boyd et al (2012), Trends in Genetics, Vol 29(4), 233-40; and Smith and Heese (2014), Plant Methods, 10: and 6, rolling. In addition, screening for pathogen resistance traits is known in the art. See, e.g., Gomez-Gomez and Boller (2000), Molecular Cell, 5: volume 1003-11; and Steuernagl et al (2016), Nature Biotechnology (Nature Biotechnology), Vol.34 (6), 652-.

Outputting the at least one biological cell. After the incubation step is complete, the at least one biological cell or cell colony can be exported outside of the growth chamber or isolated region thereof. The output may comprise using a sufficiently strong Dielectrophoretic (DEP) force to move the one or more biological cells/cell colonies. DEP forces can be optically or electronically actuated. For example, a microfluidic device may include a substrate having a DEP configuration, e.g., an optoelectronic tweezers (OET) configuration. In other embodiments, the at least one biological cell or cell colony may be output from the growth chamber or isolation region using fluid flow and/or gravity. In other embodiments, the at least one biological cell or cell colony may be output from the growth chamber or isolated region using a compressive force on the deformable cover region over the growth chamber or isolated region thereof, thereby causing localized flow of fluid out of the growth chamber or isolated region.

After the at least one biological cell or cell colony is output from the growth chamber or isolation region, the cell can be output from the flow region (e.g., channel) out of the microfluidic device. In some embodiments, outputting the cells from the flow region comprises using a strong enough DEP force to move the one or more biological cells/cell colonies. As described above, DEP forces can be generated. In some other embodiments, outputting the cell from the flow region out of the microfluidic device comprises moving the cell using fluid flow and/or gravity.

During the outputting step, an image of the at least one growth chamber and any cells contained therein may be monitored.

Conditioning at least one surface. In some embodiments, the microfluidic device is provided with at least one surface of at least one growth chamber in a conditioned state. In other embodiments, the surface of the at least one growth chamber is conditioned prior to introducing the at least one biological cell (e.g., plant protoplasts), and can be performed as part of a method of culturing the one or more biological cells. Conditioning the surface may include treating the surface with a conditioning agent (e.g., a polymer).

In some embodiments, a method for processing at least one surface of at least one growth chamber of a microfluidic device (100, 300, 400, 500A-E, and 600) is provided, the method comprising the steps of: flowing a fluid medium comprising an excess of conditioning agent into a flow channel (fig. 1A-1C, 2, 3, 4A-C); incubating the microfluidic device for a selected period of time; and replacing the media in the channel. In other embodiments, a method for priming a microfluidic device comprises the steps of: flowing a priming solution comprising a conditioning agent into the flow channel; incubating the device for a selected period of time, thereby conditioning at least one surface of the growth chamber; and replacing the solution in the channel with a fluid medium. The priming solution may comprise any of the fluid media described herein. The fluid medium of the replacement conditioning solution or fluid medium with excess conditioning agent may be any medium described herein, and may additionally comprise cells.

In some embodiments, at least one surface may be treated with a polymeric conditioning agent comprising alkylene ether moieties. The polymeric conditioning agent having alkylene ether moieties can comprise any suitable alkylene ether-containing polymer, including but not limited to any of the alkylene ether-containing polymers described above. In one embodiment, the surface of the growth chamber may be treated with an amphiphilic nonionic block copolymer comprising blocks of polyethylene oxide (PEO) and polypropylene oxide (PPO) subunits at different ratios and locations within the polymer chain (e.g.,a polymer). Specific for producing conditioned surfacesPolymers include L44, L64, P85, F68, and F127 (including F127 NF).

In other embodiments, the surface may be treated with a polymeric conditioning agent comprising carboxyl moieties. Non-limiting examples of suitable carboxylic acid-containing polymeric conditioning agents are discussed above, and thus any suitable carboxylic acid-containing polymeric conditioning agent can be used to treat a surface.

In other embodiments, the surface may be treated with a polymeric conditioning agent comprising a sugar moiety. Non-limiting examples of suitable sugar-containing polymer conditioning agents are discussed above, and thus any suitable sugar-containing polymer conditioning agent can be used to treat a surface.

In other embodiments, the surface may be treated with a polymeric conditioning agent comprising sulfonic acid moieties. Non-limiting examples of suitable sulfonic acid-containing polymeric conditioning agents are discussed above, and thus any suitable sulfonic acid-containing polymeric conditioning agent can be used to treat a surface.

In other embodiments, the surface may be treated with a polymeric conditioning agent comprising an amino acid moiety. Non-limiting examples of suitable amino acid-containing polymeric conditioning agents are discussed above, and thus any suitable amino acid-containing polymeric conditioning agent can be used to treat a surface. The polymeric amino acid-containing conditioning agent can include a protein.

In other embodiments, the surface may be treated with a polymeric conditioning agent comprising a nucleic acid moiety. Non-limiting examples of suitable nucleic acid-containing polymeric conditioning agents are discussed above, and thus any suitable nucleic acid-containing polymeric conditioning agent can be used to treat a surface.

In some embodiments, a mixture of more than one polymeric conditioning agent may be used to treat the surface of the growth chamber.

In other embodiments, conditioning comprises heating the surface of the growth chamber to a temperature of about 30 ℃. In some embodiments, the method includes heating the surface to a temperature of at least about 20 ℃, 21 ℃, 22 ℃, 23 ℃, 24 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, or about 35 ℃. In some embodiments, the method includes heating the surface to a temperature of about 25 ℃. In other embodiments, the method includes heating the surface to a temperature in a range from about 20-30 ℃, about 22 ℃ to about 28 ℃, or about 24 ℃ to about 26 ℃. In some embodiments, the method includes heating the surface to a temperature of at least about 22 ℃. In some embodiments, heating the surface comprises at least one surface conditioned by treating the surface with a polymer.

A clonal population. The methods described herein also include methods in which only one biological cell (e.g., a plant protoplast) is introduced into at least one growth chamber. A method is provided for cloning biological cells in a system comprising a microfluidic device having a flow region configured to accommodate flow of a first fluidic medium; and at least one growth chamber comprising an isolation region and a communication region, the isolation region in fluid communication with the communication region, and the communication region comprising a proximal opening to the flow region, the method comprising the steps of: introducing biological cells into at least one growth chamber, wherein the at least one growth chamber is configured with at least one surface conditioned to support cell growth, viability, portability, or any combination thereof; and incubating the biological cells for at least a period of time sufficient to expand the biological cells to produce a clonal population of biological cells. In some embodiments, the system may be any system described herein. The microfluidic device may be any of the microfluidic devices described herein.

In some embodiments of methods of cloning biological cells, at least one conditioned surface may comprise a linking group covalently attached to the surface, and the linking group may be attached to a moiety configured to support cell growth, viability, or portability of one or more biological cells within a microfluidic device. In some embodiments, the linking group can include a siloxy linking group. In other embodiments, the linking group can include a phosphonate linking group. In some embodiments, the linking group can be indirectly attached to a moiety configured to support cell growth, viability, portability, or any combination thereof. In other embodiments, the linking group may be directly attached to a moiety configured to support cell growth, viability, portability, or any combination thereof. The linking group can be indirectly attached to the moiety configured to support cell growth, viability, or mobility via a connection to the linker. In some embodiments, the linking group can be indirectly connected to the moiety configured to support cell growth, viability, or mobility via a connection to the first end of the linker. In some embodiments, the linker may further comprise a linear moiety, wherein the backbone of the linear moiety comprises from 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms. In some embodiments, the backbone of the linear moiety may include one or more arylene moieties. In other embodiments, the linker may include a triazolylene moiety. In some embodiments, the triazolylene moiety may interrupt a linear portion of the linker or may be attached to a linear portion of the linker at the second end. In various embodiments, the moiety configured to support cell growth and/or viability and/or portability may comprise an alkyl or fluoroalkyl (including perfluoroalkyl) moiety; monosaccharides or polysaccharides (which may include but are not limited to dextran); alcohols (including but not limited to propargyl alcohol); polyols, including but not limited to polyvinyl alcohol; alkylene ethers including, but not limited to, polyethylene glycol; polyelectrolytes (including but not limited to polyacrylic acid or polyvinylphosphonic acid); amino (including derivatives thereof such as, but not limited to, alkylated amines, hydroxyalkylated amino, guanidino, and heterocyclic groups containing nitrogen ring atoms that are not aromatic, such as, but not limited to morpholinyl or piperazinyl); carboxylic acids, including but not limited to propiolic acid (which can provide a carboxylate anionic surface); phosphonic acids, including but not limited to ethynylphosphonic acid (which can provide a phosphonate anionic surface); a sulfonate anion; a carboxybetaine; a sulfobetaine; sulfamic acid; or an amino acid. In some embodiments, at least one conditioned surface comprises an alkyl or perfluoroalkyl moiety. In other embodiments, at least one conditioned surface comprises an alkylene ether moiety or a dextran moiety.

In various embodiments, the method may further comprise the step of conditioning at least one surface of at least one growth chamber. In some embodiments, conditioning comprises treating at least one surface with one or more agents that support cell portability within the microfluidic device. In some embodiments, conditioning may comprise treating at least one surface of at least one growth chamber with a conditioning agent comprising a polymer. In some embodiments, the polymer may include alkylene ether moieties. In some embodiments, the polymer may include carboxylic acid moieties. In some embodiments, the polymer can include a sugar moiety. In other embodiments, the polymer may include sulfonic acid moieties. In other embodiments, the polymer may include an amino acid moiety. In other embodiments, the polymer can include a nucleic acid moiety.

In various embodiments, conditioning may comprise heating at least one surface of at least one growth chamber to a temperature of about 30 ℃.

In various embodiments, the method may further comprise the step of introducing a first fluidic medium into a microfluidic channel of a flow region of the microfluidic device. In some embodiments, the first fluid medium may be introduced prior to introduction of the biological cells (e.g., plant protoplasts). In some embodiments, introducing the biological cells into the at least one growth chamber may include moving the biological cells using a Dielectrophoretic (DEP) force of sufficient strength. In some embodiments, the DEP force may be optically actuated. In some embodiments, DEP forces may be generated by optoelectronic tweezers (OET). In some other embodiments, introducing the biological cells into the at least one growth chamber may comprise using fluid flow and/or gravity.

In some embodiments, introducing biological cells into the at least one growth chamber can further comprise introducing biological cells into the isolated region of the at least one growth chamber. In some embodiments, the isolated region of at least one growth chamber can have a volume sufficient to support cell expansion to no more than 1 x 10²Size of individual cells. In some embodiments, the isolation region may be at least substantially filled with the second fluid medium. In some embodiments, the flow region may be in fluid communication with the proximal opening of the communication region of the at least one growth chamber, and further wherein the communication region may also be in fluid communication with the isolation region of the growth chamber.

In various embodiments, the method may further comprise the step of perfusing the first fluidic medium during the incubating step, wherein the first fluidic medium may be introduced via at least one inlet port of the microfluidic device, and wherein the first fluidic medium (optionally including components from the second fluidic medium) may be output via at least one outlet port of the microfluidic device. In some embodiments, the perfusion may be discontinuous. In some other embodiments, the perfusion may be periodic. In other embodiments, the perfusion may be aperiodic. In some embodiments, the perfusion of the first fluid medium may be sufficient to allow diffusion of a component of the second fluid medium in the isolation region into the first fluid medium in the flow region and/or diffusion of a component of the first fluid medium into the second fluid medium in the isolation region; and the first medium may not substantially flow into the isolation zone. In some embodiments, the perfusion of the first fluidic medium may be performed for a duration of about 45 seconds to about 90 seconds about every 10 minutes to about every 30 minutes. In some embodiments, the perfusion of the first fluid medium may be performed for a duration of about 2 hours to about 4 hours. In some embodiments, the at least one biological cell may be incubated for a period of time of about 1 day to about 14 days, or longer.

In some embodiments, the components of the first fluid medium may include a liquid component and a gaseous component. In various embodiments, the method may further comprise the steps of: the first fluid medium is saturated by dissolved gaseous molecules before being introduced into the microfluidic device. In various embodiments, the method may further comprise the steps of: the microfluidic device is brought into contact with a gaseous environment capable of saturating the first fluid medium or the second fluid medium with dissolved gaseous molecules. In various embodiments, the method may further comprise the steps of: the pH of the first fluid medium is adjusted while introducing the dissolved gaseous molecules. In some embodiments, saturating the first fluidic medium with the gaseous molecules may be performed in the reservoir via the inlet port, in a gas-permeable connector between the reservoir and the inlet port, or via a gas-permeable portion of a lid of the microfluidic device. In some embodiments, the component of the first fluid medium may comprise at least one secreted component from a feeder cell culture.

In various embodiments, the method may further comprise the step of detecting the pH of the first fluid medium as it is output via the at least one outlet. In some embodiments, the detecting step may be performed at a location proximate to the at least one outlet. In various embodiments, the method may further comprise the step of detecting the pH of the first fluid medium when the first fluid medium is introduced via the at least one inlet. In some embodiments, the sensor may be an optical sensor. In various embodiments, the method may further comprise the step of altering the composition of the first fluid medium.

In various embodiments, the method may further comprise the step of monitoring an image of the at least one growth chamber and any cells contained therein.

In various embodiments, the biological cell can be a plant cell, such as a protoplast. The plant may be any type of plant, such as a plant used in agriculture, non-limiting examples of which include lettuce, tomato, corn, wheat, tobacco, and the like.

In some embodiments, the biological cell may be a plurality of biological cells and the at least one growth chamber is a plurality of growth chambers. In various embodiments, the method may further comprise the steps of: no more than one of the plurality of biological cells is moved into each of the plurality of growth chambers.

In some embodiments of the method of cloning a biological cell, the conditioned surface may further comprise a cleavable (cleavable) moiety. The method may include the step of disrupting the cleavable moiety prior to exporting one or more clonal populations of biological cells from the growth chamber or an isolation region thereof.

In various embodiments, the method can further comprise the step of exporting the one or more biological cells of the clonal population outside the growth chamber or the isolation region thereof. In some embodiments, the outputting may include using a sufficiently strong Dielectrophoretic (DEP) force to move the one or more biological cells. In some embodiments, the DEP force is optically actuated. In some embodiments, DEP forces may be generated by optoelectronic tweezers (OET). In some embodiments, outputting may include using fluid flow and/or gravity. In some embodiments, the output may include using a compressive force on a deformable cover region over the growth chamber or an isolated region thereof. In various embodiments, the method can further include the step of outputting one or more biological cells of the clonal population from the flow region to outside the microfluidic device. In some embodiments, outputting may include moving the one or more biological cells using a sufficiently strong DEP force. In some embodiments, the DEP force is optically actuated. In some embodiments, DEP forces may be generated by optoelectronic tweezers (OET). In some embodiments, outputting may include using fluid flow and/or gravity.

A kit. Kits may be provided for culturing and screening plant cells, particularly plant protoplasts, wherein the kit comprises: a microfluidic device having a flow region configured to accommodate flow of a first fluidic medium and at least one growth chamber; a surface conditioner; and a measuring agent. In this embodiment, the at least one growth chamber is not pre-treated to condition at least one surface of the at least one growth chamber, and the conditioned surface is produced by treatment with a surface conditioner prior to introduction into the cells. Other kits for culturing plant cells (e.g., plant protoplasts) are also provided, wherein the kit comprises: a microfluidic device having a flow region configured to accommodate flow of a first fluidic medium and at least one growth chamber comprising an isolation region and a communication region, wherein the isolation region is in fluid communication with the communication region and the communication region comprises a proximal opening to the flow region; a surface conditioner; and an assay agent, wherein the surface conditioning agent, when applied to an interior surface of the microfluidic device, produces a surface that supports cell growth, viability, portability, or any combination thereof. Other kits for culturing and screening plant cells (e.g., plant protoplasts) are also provided, comprising: a microfluidic device comprising a flow region configured to accommodate flow of a first fluidic medium and at least one growth chamber comprising an isolation region and a communication region, wherein the isolation region is in fluid communication with the communication region and the communication region comprises a proximal opening to the flow region, and the at least one growth chamber has at least one surface comprising a covalently bound surface modifying ligand; and a surface. Alternatively, a kit for culturing biological cells may be provided, wherein the kit comprises: a microfluidic device having a flow region configured to accommodate a flow of a first fluidic medium and at least one growth chamber having at least one conditioned surface, wherein the at least one conditioned surface can support cell growth, viability, portability, or any combination thereof; and a surface conditioner. The microfluidic device of any of the kits may be any of the microfluidic devices 100, 200, 240, 290, 400, 500A-E, or 600, and have any of the features described above.

The microfluidic device of any of the kits may further comprise a microfluidic channel comprising at least a portion of the flow region, and the device may further comprise a growth chamber having a communication region directly leading to the microfluidic channel. The growth chamber may also include an isolation region. The isolation region may be in fluid communication with the communication region and may be configured to contain a second fluid medium, wherein when the flow region and the at least one growth chamber are substantially filled with the first fluid medium and the second fluid medium, respectively, then a component of the second fluid medium diffuses into the first fluid medium and/or a component of the first fluid medium diffuses into the second fluid medium; the first medium does not substantially flow into the isolation zone.

In various embodiments of any of the kits, the growth chamber can be configured similar to growth chambers 124, 126, 128, 130, 244, 246, 248, or 436 of fig. 1A-1C, 2, 3, and 4A-4C, wherein the volume of the isolation region of the growth chamber can be configured to support no more than about 1x10³、5x10²、4x10²、3x10²、2x10²、1x10²50, 25, 15 or 10 cells. In other embodiments, the isolated region of the growth chamber has a shape that can support up to about 10, 50, or 1x10²Volume of individual cells. Any configuration of growth chambers as described above may be used in the growth chambers of the microfluidic devices of the kits.

In various embodiments of any of the kits, the growth chamber can be configured to maintain a size of no more than 1x10²A biological cell, wherein the volume of the growth chamber can not exceed 1x10⁷Cubic microns. In other embodiments, wherein no more than 1 × 10 may be maintained²The volume of the biological cell and the growth chamber can not exceed 5 x10⁶Cubic microns. In thatIn other embodiments, no more than 50 biological cells may be maintained, and the volume of the growth chamber may be no more than 1 × 10⁶Cubic micron, or not more than 5 × 10⁵Cubic microns. In the kit, the microfluidic device may have any number of growth chambers as described above.

The microfluidic device of any of the kits can further comprise at least one inlet port configured to input fluidic media (e.g., first fluidic media or second fluidic media) into the flow region and at least one outlet port configured to receive fluidic media (e.g., spent first fluidic media) upon exit of the fluidic media from the flow region.

The microfluidic device of any of the kits can further comprise a substrate having a plurality of DEP electrodes, wherein a surface of the substrate forms a surface of the growth chamber and the flow region. The plurality of DEP electrodes can be configured to generate a sufficiently strong Dielectrophoretic (DEP) force to move one or more biological cells (e.g., clonal populations) into the growth chamber or isolated region thereof or move one or more cells in a biological cell culture out of the growth chamber or isolated region thereof. DEP electrodes, and thus DEP forces, can be optically actuated. Such optically actuated DEP electrodes may be dummy electrodes (e.g., regions of an amorphous silicon substrate having increased conductivity due to incident light), phototransistors, or electrodes that are turned on or off by corresponding phototransistors. Alternatively, the DEP electrode, and thus the DEP force, may be electrically actuated. In some other embodiments, the microfluidic device may further include a substrate having a plurality of transistors, wherein a surface of the substrate forms a surface of the growth chamber and the flow region. The plurality of transistors are capable of generating a sufficiently strong Dielectrophoretic (DEP) force to introduce the biological cell or to move one or more cells in the biological cell culture out of the growth chamber or isolated region thereof. Each of the plurality of transistors may be optically actuated, and the DEP force may be generated by the optoelectronic tweezers.

The microfluidic device of any of the kits can further comprise a deformable cover region over the at least one growth chamber or the isolation region thereof, whereby depression of the deformable cover region applies a force to export one or more biological cells (e.g., clonal population) from the growth region to the flow region.

The microfluidic device of any of the kits may be configured with a substantially gas-impermeable cover. Alternatively, all portions of the lid may be configured to be gas permeable. The permeable portion of the lid may be permeable to at least one of carbon dioxide, oxygen, and nitrogen. In some embodiments, the lid (or a portion thereof) may be permeable to a combination of more than one of carbon dioxide, oxygen, or nitrogen.

Any kit may further include a reservoir configured to contain a fluidic medium. The reservoir may be in fluid communication with any of the microfluidic devices described herein. The reservoir may be configured such that the fluid medium present in the reservoir may be in contact with a gaseous environment capable of saturating the fluid medium with dissolved gaseous molecules. The reservoir may also be configured to hold a population of feeder cells in fluid contact with the fluid medium.

Any kit may include at least one connecting conduit configured to connect to an inlet port and/or an outlet port of a microfluidic device. The connecting conduit may also be configured to connect to a reservoir or flow controller (e.g., a pump assembly). The connecting conduit may be gas permeable. The gas permeable connecting conduit may be permeable to at least one of carbon dioxide, oxygen and nitrogen. In some embodiments, the gas permeable conduit may be permeable to a combination of more than one of carbon dioxide, oxygen, or nitrogen.

Any of the kits may further comprise a sensor configured to detect the pH of the first fluid medium. The sensor may be connected to (or connectable to) an inlet of the microfluidic device or a connection conduit attached thereto. Alternatively, the sensor may be integrated with the microfluidic device. The sensor may be connected proximal to the point at which the fluidic medium enters the microfluidic device. The kit may include a sensor configured to detect a pH of the fluid medium at the outlet of the microfluidic device. The sensor may be connected to (or connectable to) an outlet of the microfluidic device or a connection conduit attached thereto. Alternatively, the sensor may be integrated with the microfluidic device. The sensor may be connected proximal to the point at which the fluidic medium enters the microfluidic device. The kit may include a sensor configured to detect a pH of the fluid medium at the outlet of the microfluidic device. The sensor may be connected to (or connectable to) an outlet of the microfluidic device or a connection conduit attached thereto. Alternatively, the sensor may be integrated with the microfluidic device. The sensor may be connected to the proximal end of the point where the fluid medium exits the microfluidic device. The sensor, whether attached to the inlet and/or outlet of the microfluidic device, may be an optical sensor. The optical sensor may include an LED and an integrated colorimetric sensor, which may optionally be a color sensitive phototransistor. The kit may also include drive electronics to control the pH sensor and receive output therefrom. The kit may also include a pH detection reagent. The pH detection reagent may be a pH sensitive dye that can detect under visible light.

Any kit may also include a culture medium having components capable of enhancing the viability of biological cells on the microfluidic device. These components may be any suitable culture medium components known in the art, including any of the components discussed above with respect to the fluid medium components.

Any kit may also include at least one reagent to detect the status of a biological cell or cell population. Reagents configured to detect the state of a cell are well known in the art and may be used, for example, to detect whether a cell is viable or dead; screening for substances of interest such as antibodies, cytokines or growth factors; or with a cell surface marker of interest. Such reagents may be used in the kits and methods described herein without limitation.

For any of the kits provided herein, the components of the kit can be in separate containers. For any component of the kit provided in solution, the component can be present at a concentration of about 1X, 5X, 10X, 100X, or about 1000X of the concentration used in the methods of the present disclosure.

Preconditioning at least one growth chamber of a microfluidic device to prevent degradation of the microfluidic deviceA kit for conditioning at least one surface of at least one growth chamber and producing a conditioned surface by treatment with a surface conditioner, or for a kit comprising a microfluidic device having a flow region configured to accommodate flow of a first fluid medium and at least one growth chamber having at least one conditioned surface that can support cell growth, viability, portability, or any combination thereof, and a surface conditioner, the surface of the growth chamber can be preconditioned by the surface conditioner. The surface conditioning agent may comprise a polymer, which may be any one or more of the polymers described above for use as surface conditioning agents. In some embodiments, the surface conditioning agent can include a polymer having alkylene ether moieties, carboxylic acid moieties, sulfonic acid moieties, amino acid moieties, nucleic acid moieties, sugar moieties, or any combination thereof. The surface conditioning agent may comprise a PEO-PPO block copolymer, for example, A polymer (e.g., L44, L64, P85, or F127).

Alternatively, a surface conditioning agent for conditioning the growth chamber surface may be included in the kit, separate from the microfluidic device. In other embodiments of the kit, the pre-conditioned microfluidic device is included with a surface conditioning agent that is different from the surface conditioning agent used to condition the surface of the growth chamber. The different surface conditioning agent may be any of the surface conditioning agents discussed above. In some embodiments, more than one surface conditioning agent is included in the kit.

In various embodiments of kits having a microfluidic device in which at least one growth chamber of the microfluidic device has not been pretreated to condition at least one surface, the kit can further comprise a culture medium suitable for culturing one or more biological cells. In some embodiments, the kit may further include a culture medium additive comprising an agent capable of supplementing the conditioning of the growth chamber surface. The culture medium additive may include a conditioner as described above or another chemical that enhances the ability of at least one surface of at least one growth chamber to support cell growth, viability, portability, or any combination thereof. This may include growth factors, hormones, antioxidants, or vitamins, among others.

The kit may further comprise a flow controller configured to perfuse the at least first fluidic medium, which flow controller may be a separate component of the microfluidic device or may be incorporated as part of the microfluidic device. The controller may be configured to discontinuously perfuse the fluid medium. Thus, the controller may be configured to perfuse the fluid medium in a periodic or aperiodic manner.

In another aspect, a kit for culturing biological cells (e.g., plant protoplasts) is provided, the kit comprising: a microfluidic device having a flow region configured to accommodate flow of a first fluidic medium and at least one growth chamber comprising an isolation region and a communication region, wherein the isolation region is in fluid communication with the communication region and the communication region comprises a proximal opening to the flow region; further wherein at least one growth chamber comprises at least one conditioned surface for cell growth, viability, portability, or any combination thereof. The microfluidic device may be any microfluidic device described herein, and may have any growth chamber described herein. The microfluidic device can have a substrate with any kind of DEP configuration described herein. Alternatively, the DEP configuration may be optically actuated. The substrate of the microfluidic device may have a surface comprising a substrate composition of formula 1 or formula 2 as described herein, and have all the features as described above.

The at least one conditioned surface of the microfluidic device of the kit can include a sugar moiety, an alkylene ether moiety, an amino acid moiety, an alkyl moiety, a fluoroalkyl moiety (which can include a perfluoroalkyl moiety), an anionic moiety, a cationic moiety, and/or a zwitterionic moiety. In some embodiments, the conditioned surface of the microfluidic device can include a sugar moiety, an alkylene ether moiety, an alkyl moiety, a fluoroalkyl moiety, or an amino acid moiety. The alkyl or perfluoroalkyl moiety may have a backbone length of greater than 10 carbons. In some embodiments, the conditioned surface that supports cell growth, viability, transplantability, or any combination thereof, may include alkyl or fluoroalkyl (including perfluoroalkyl) moieties; monosaccharides or polysaccharides (which may include but are not limited to dextran); alcohols (including but not limited to propargyl alcohol); polyols, including but not limited to polyvinyl alcohol; alkylene ethers including, but not limited to, polyethylene glycol; polyelectrolytes (including but not limited to polyacrylic acid or polyvinylphosphonic acid); amino (including derivatives thereof such as, but not limited to, alkylated amines, hydroxyalkylated amino, guanidino, and heterocyclic groups containing nitrogen ring atoms that are not aromatic, such as, but not limited to morpholinyl or piperazinyl); carboxylic acids, including but not limited to propiolic acid (which can provide a carboxylate anionic surface); phosphonic acids, including but not limited to ethynylphosphonic acid (which can provide a phosphonate anionic surface); a sulfonate anion; a carboxybetaine; a sulfobetaine; sulfamic acid; or an amino acid.

In some embodiments of the kit, the conditioned surface may include a linking group covalently attached to a surface of the microfluidic device, and the linking group may be attached to a moiety configured to support cell growth, viability, portability, or any combination thereof, of one or more biological cells within the microfluidic device. The linking group may be a siloxy linking group. Alternatively, the linking group may be a phosphonate linking group. In some embodiments of the kit, the linking group of the conditioned surface may be directly linked to a moiety configured to support cell growth, viability, portability, or any combination thereof.

In other embodiments, the linking group may be indirectly attached via a linker to a moiety configured to support cell growth, viability, portability, or any combination thereof. The linking group can be indirectly connected to the moiety configured to support cell growth, viability, portability, or any combination thereof via a connection to the first end of the linker. The linker may also include a linear moiety, wherein the backbone of the linear moiety comprises 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms. In some embodiments of the kit, the linker of the conditioned surface further comprises a triazolylene moiety. The cleavable moiety is configured to allow disruption of the conditioned surface, thereby facilitating the portability of the biological cell. The kit also includes a reagent configured to cleave the cleavable moiety of the conditioned surface.

In various embodiments of the kit, the kit can further comprise a surface conditioning agent. In some embodiments, the surface conditioning agent can comprise a polymer comprising at least one of an alkylene ether moiety, a carboxylic acid moiety, a sulfonic acid moiety, a phosphonic acid moiety, an amino acid moiety, a nucleic acid moiety, or a sugar moiety. In some other embodiments, the surface conditioning agent comprises a polymer comprising at least one of an alkylene ether moiety, an amino acid moiety, or a sugar moiety. In some other embodiments, the conditioned surface may comprise a cleavable moiety.

In other embodiments of the kit, the surface conditioning agent comprises at least one cell adhesion blocking molecule. In some embodiments, the at least one cell adhesion blocking molecule can disrupt actin filament formation, block integrin receptors, or reduce cell binding to DNA-fouled surfaces. In some embodiments, the at least one cell adhesion blocking molecule may be cytochalasin B, an RGD-containing peptide, a DNase1 protein, a fibronectin inhibitor, or an antibody to an integrin. In some embodiments, the at least one cell adhesion blocking molecule may comprise a combination of more than one type of cell adhesion blocking molecule.

In various embodiments of the kit, the kit can further comprise a culture medium suitable for culturing one or more biological cells. In some embodiments, the kit may include a culture medium additive comprising a reagent configured to supplement conditioning of at least one surface of the growth chamber. The culture medium additive may include a conditioner as described above or another chemical that enhances the ability of at least one surface of at least one growth chamber to support cell growth, viability, portability, or any combination thereof. This may include growth factors, hormones, antioxidants, or vitamins, among others.

In various embodiments of the kit, the kit can include at least one reagent for detecting the status of one or more biological cells.

In another aspect, a kit for culturing biological cells includes a microfluidic device for culturing one or more biological cells, the microfluidic device including a flow region configured to accommodate a flow of a first fluidic medium; and at least one growth chamber comprising an isolation region and a communication region, wherein the isolation region is in fluid communication with the communication region, and the communication region has a proximal opening to the flow region; and at least one growth chamber has at least one surface with surface-modifying ligands. The microfluidic device may be any of the microfluidic devices described herein. The surface may include a substrate having a Dielectrophoresis (DEP) configuration. The DEP configuration can be any DEP configuration described herein. The DEP configuration may be optically actuated. The substrate is any substrate having a surface modifying ligand described herein, and may have the structure of formula 3, and may include all of the features described above:

In various embodiments of kits having a microfluidic device with at least one surface comprising a surface modifying ligand, the surface modifying ligand can be covalently attached to an oxide moiety of the surface of the substrate. The surface modifying ligand may include a reactive moiety. The reactive moiety of the surface modifying ligand may be an azido, amino, bromo, thiol, activated ester, succinimidyl, or alkynyl moiety. The surface-modifying ligand may be covalently attached to the oxide moiety via a linking group. In some embodiments, the linking group can be a siloxy moiety. In other embodiments, the linking group may be a phosphonate moiety. The linking group may be indirectly linked to the reactive moiety of the surface modifying ligand via a linker. The linker may comprise a linear moiety, wherein the backbone of the linear moiety comprises from 1 to 100 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms. In some embodiments, the surface modifying ligand may comprise one or more cleavable moieties. The one or more cleavable moieties may be configured to allow for the disruption of the conditioned surface of the microfluidic device once formed, thereby facilitating the portability of the one or more biological cells after culture.

In some embodiments of a kit having a microfluidic device having at least one surface comprising a surface modifying ligand, the kit may further comprise a conditioning modifier comprising a first portion configured to support cell growth, viability, portability, or any combination thereof, and a second portion configured to react with a reactive portion of the surface modifying ligand, which may have the structure of formula 5, and have any of the features described herein:

the second portion may be configured to transform the surface-modified ligand into a conditioned surface configured to support cell growth, viability, portability, or any combination thereof of one or more biological cells within the growth chamber upon reaction with a reactive portion of the surface-modified ligand of the microfluidic device of the kit. The first portion may include an alkylene oxide moiety, a sugar moiety; an alkyl moiety, a perfluoroalkyl moiety, an amino acid moiety, an anionic moiety, a cationic moiety, or a zwitterionic moiety. In some embodiments, the first portion may comprise an alkyl or fluoroalkyl (including perfluoroalkyl) moiety; monosaccharides or polysaccharides (which may include but are not limited to dextran); alcohols (including but not limited to propargyl alcohol); polyols, including but not limited to polyvinyl alcohol; alkylene ethers including, but not limited to, polyethylene glycol; polyelectrolytes (including but not limited to polyacrylic acid or polyvinylphosphonic acid); amino (including derivatives thereof such as, but not limited to, alkylated amines, hydroxyalkylated amino, guanidino, and heterocyclic groups containing nitrogen ring atoms that are not aromatic, such as, but not limited to morpholinyl or piperazinyl); carboxylic acids, including but not limited to propiolic acid (which can provide a carboxylate anionic surface); phosphonic acids, including but not limited to ethynylphosphonic acid (which can provide a phosphonate anionic surface); a sulfonate anion; a carboxybetaine; a sulfobetaine; sulfamic acid; or an amino acid. The second moiety may be an amino, carboxylic acid, alkyne, azide, aldehyde, bromide, or thiol moiety. In some embodiments, the first portion of the conditioning modifier or linker L' (as described above for formula 5) can comprise a cleavable moiety. The cleavable moiety may be configured to allow disruption of the conditioned surface, thereby facilitating the portability of the biological cell. In some embodiments, the kit can further include a reagent configured to lyse the cleavable moiety of the conditioned surface.

In some embodiments of kits having a microfluidic device with at least one surface comprising a surface modifying ligand, the kit may further comprise a surface conditioning agent.

In some embodiments of kits having a microfluidic device with at least one surface comprising a surface modifying ligand, the surface conditioning agent can comprise a polymer comprising at least one of an alkylene ether moiety, a carboxylic acid moiety, a sulfonic acid moiety, a phosphonic acid moiety, an amino acid moiety, a nucleic acid moiety, or a sugar moiety. In some other embodiments, the surface conditioning agent comprises a polymer comprising at least one of an alkylene ether moiety, an amino acid moiety, or a sugar moiety. In some other embodiments, the conditioned surface may comprise a cleavable moiety.

In some embodiments of kits having a microfluidic device with at least one surface comprising a surface modifying ligand, the surface conditioning agent comprises at least one cell adhesion blocking molecule. In some embodiments, the at least one cell adhesion blocking molecule can disrupt actin filament formation, block integrin receptors, or reduce cell binding to DNA-fouled surfaces. In some embodiments, the at least one cell adhesion blocking molecule may be cytochalasin B, an RGD-containing peptide, a DNase 1 protein, a fibronectin inhibitor, or an antibody to an integrin. In some embodiments, the at least one cell adhesion blocking molecule may comprise a combination of more than one type of cell adhesion blocking molecule.

In some embodiments of kits having a microfluidic device with at least one surface comprising a surface-modified ligand, the kit may further comprise a culture medium suitable for culturing one or more biological cells. In some embodiments, the kit may further include a culture medium additive comprising a reagent configured to supplement conditioning of at least one surface of the growth chamber. The culture medium additive may include a conditioner as described above or another chemical that enhances the ability of at least one surface of at least one growth chamber to support cell growth, viability, portability, or any combination thereof. This may include growth factors, hormones, antioxidants, or vitamins, among others.

In some embodiments of kits having a microfluidic device with at least one surface comprising a surface-modified ligand, the kit can further comprise at least one reagent for detecting a state of one or more biological cells.

Examples of the invention

Example 1 culture and growth of grape and lettuce protoplasts in a microfluidic device.

Systems and microfluidic devices: the system comprisesInstrument (Berkeley Lights, Inc.) and OptoSelect^TM3500 and 1750 microfluidic chips (Berkeley Lights, Inc). The instrument includes a flow controller, a temperature controller, a fluid medium conditioning and pump assembly, a structured light source for light activated DEP configuration, a mounting stage/mount and a camera. The microfluidic chip includes a substrate having an array of phototransistors resting on a first electrode and a lid having an ITO electrode on its inner surface; the silicone microfluidic circuit material is sandwiched between and cooperates with the substrate and the cover to define a microfluidic circuit comprising an inlet, an outlet, and a plurality of microfluidic channels. OptoSelect ^TM3500 chip includes about 3500 isolation enclosures each having a volume of about 5x10⁵Cubic microns (i.e., -0.5 nL); OptoSelect^TM1750 the chip included about 1750 isolated pens, the volume of each penAbout 1.1x10⁶Cubic microns (i.e., -1.1 nL). The interior surface of the microfluidic chip includes a coating of covalently attached Polyethylene (PEG) polymer.

First, grape protoplasts were prepared according to standard procedures and loaded into OptoSelect in standard protoplast media^TM3500 microfluidic chip, which is introduced into a growth chamber (in this case, an isolation pen) using gravity (i.e., by standing the microfluidic chip upright on its side for a period of time), then incubated (i.e., incubated) in standard protoplast medium for a period of about 48 hours, and subjected to continuous perfusion. Protoplasts show sustained viability during the course of the experiment, as determined by time-lapse imaging, which shows continuous movement of internal structures in the protoplasts. Figure 8 shows bright field images of grape protoplasts collected during the experiment, showing the presence of 1-3 protoplasts per isolation pen. As an alternative to gravity loading, DEP forces (e.g., optically activated DEP or OEP) may be used ^TM) Grape protoplasts were loaded into isolation pens.

Next, lettuce protoplasts were prepared according to standard procedures and loaded into OptoSelect in standard protoplast media^TM1750 in a microfluidic chip, it is introduced into a growth chamber (in this case, an isolation pen) using gravity (i.e., by standing the microfluidic chip upright on its side for a period of time), then incubated in standard protoplast media for a period of about 14 days, and perfused intermittently with fresh protoplast media (including fluorescently labeled dyes as described below) every three days. During the incubation period, lettuce protoplasts were stained with various dyes, including (i) fluorescein diacetate for cell viability, (ii) chlorophyll stain, (iii) fluorescent whitening agent for cell wall detection, and (iv) Hoechst (Hoechst) for cell nucleus detection. Fig. 9 shows exemplary images of two different isolation pens containing lettuce protoplasts at the end of the fourteen day incubation period, including bright field and fluorescence images of protoplasts stained with Hoechst, chlorophyll stain, or images with a combination of Hoechst and chlorophyll stain. Fluorescence as expected Images of the biotin and fluorescent brightener stains (not shown) reveal that cell wall reconstruction correlates with viability.

As an alternative to gravity loading, experiments with lettuce protoplasts were performed in which DEP forces (e.g., light activated DEP or OEP) were used^TM) Protoplasts were loaded into isolation pens. Very high percent fencing was achieved using the standard DEP force settings for mammalian cells (>90%). Furthermore, the viability of DEP-fenced lettuce protoplasts showed no significant change relative to gravity-loaded protoplasts.

During the fourteen day culture period, lettuce protoplasts begin to adhere to the surface of the isolation pens. To derive and recover protoplasts from the microfluidic chip, a gentle laser treatment (i.e., 40% power for 400 milliseconds) was applied to the surface of the substrate in the upper right corner of the isolation pen. Laser treatment shed the protoplasts sufficiently to allow DEP force to be used to selectively export and recover protoplast clones from the chip. After export, the protoplast clones can be treated by standard methods to regenerate whole plants.

Example 2 genotyping protoplasts

Plant protoplasts are cultured in a microfluidic device to generate clonal colonies, essentially as described in example 1 above. The protoplasts can be grape protoplasts, lettuce protoplasts, or any other plant protoplasts described herein. After the formation of colonies, cellulase enzymes are perfused with fresh culture medium and then incubated for a time sufficient to allow the protoplasts to separate from each other (which can be visually monitored to confirm separation). For each of one or more selected protoplast colonies (e.g., selected based on viability markers and/or appearance), a subset of the protoplasts in the colony are individually output from their corresponding sequestration pens using DEP force, optionally by applying laser pulses. The exported cells are then recovered off-chip in standard tissue culture plates by flowing the medium containing the exported protoplasts out of the microfluidic chip. The output protoplasts are processed to obtain nucleic acids (e.g., RNA for transcriptome analysis and/or DNA for genomic analysis), sequenced, and used to genotype live protoplasts from colonies remaining on the chip. Fig. 10 provides a schematic illustration of this workflow.

EXAMPLE 3 identification of disease resistance traits in microfluidic devices

Plant protoplasts are cultured in a microfluidic device to generate clonal colonies, essentially as described in example 1 above. The protoplasts can be grape protoplasts, lettuce protoplasts, or any other plant protoplasts described herein.

After culturing the protoplasts for the first time, exposing/contacting the protoplasts to/with the pathogen for a second time. The pathogen may be the pathogen itself, such as a virus, bacterial cell, fungal cell, and the like. Alternatively, the pathogen may be a part of a pathogen that has the ability to trigger plant immunity. For example, the pathogen can be a flagellin (e.g., a bacterial flagellin), a lipopolysaccharide (e.g., LPS a), a peptidoglycan, a chitin protein, a capsid protein (e.g., a viral capsid protein), and the like. To contact the protoplasts with the pathogen, the pathogen flows into the microfluidic device and is allowed to diffuse into the isolation pens, where it can contact the surface of the protoplasts. Alternatively, after light is emitted into the microfluidic device, the pathogen can be actively moved into the isolation pen using forces such as DEP, local flow, etc. Active movement of pathogens tends to work better with intact pathogens, while passive movement of pathogens tends to work better with molecular agents.

During the second time period, changes in protoplast viability are monitored. Viability can be monitored by bright field observation, fluorescent viability stains (e.g., fluorescein diacetate, Hoechst, fluorescent whitening agents, chlorophyll stains, etc.). If a plant protoplast is resistant to a pathogen, exposing the plant protoplast to the pathogen will induce a cell death pathway in the protoplast, resulting in a significant decrease in viability. However, if protoplasts remain viable after exposure to a pathogen, their output can be genotyped to identify genetic origins that lack pathogen resistance. Genotyping may focus on, for example, known plant immune genes, such as Effector Triggered Immunity (ETI) genes, Effector Triggered Susceptibility (ETS) genes, and/or Pathogen Associated Molecular Pattern (PAMP) genes, and optionally, known plant immune genes may be selected based on the pathogen to which the protoplast is exposed.

Prior to introducing the protoplasts into the microfluidic device, the protoplasts can be treated with a mutagen (e.g., a chemical mutagen or transfected with a nucleic acid targeting construct (e.g., a gene editing construct)). Alternatively, the protoplasts can be mutagenized on-chip by flowing the mutagen into the microfluidic device and contacting the protoplasts within the isolation pens with the mutagen (e.g., by allowing the mutagen to diffuse into the isolation pens toward the protoplasts).

FIG. 11 provides a schematic of the foregoing workflow for identifying disease resistance traits.

The examples described herein are exemplary in nature and are in no way intended to limit the scope of the methods and kits described throughout the specification.

List of embodiments

Example 1. a microfluidic device for culturing one or more plant protoplasts, the device comprising: a flow region configured to accommodate a flow of a first fluid medium; and at least one growth chamber comprising an isolation region and a communication region, the isolation region in fluid communication with the communication region and the communication region comprising a proximal opening to the flow region, wherein at least one growth chamber further comprises at least one surface conditioned to support cell growth, viability, portability, or any combination thereof in the microfluidic device.

Embodiment 2. the microfluidic device according to embodiment 1, wherein at least one conditioned surface is conditioned by one or more reagents within the microfluidic device that support cell portability.

Embodiment 3. the microfluidic device according to embodiment 1 or 2, wherein at least one conditioned surface is conditioned by a polymer comprising alkylene ether moieties.

Embodiment 4. the microfluidic device according to any one of embodiments 1-3, wherein at least one conditioned surface is conditioned by a polymer comprising a sugar moiety.

Embodiment 5. the microfluidic device according to any one of embodiments 1-4, wherein at least one conditioned surface is conditioned by a polymer comprising an amino acid moiety.

Embodiment 6. the microfluidic device according to any one of embodiments 1-5, wherein at least one conditioned surface of the microfluidic device is conditioned by a polymer comprising a carboxylic acid moiety, a sulfonic acid moiety, a nucleic acid moiety, or a phosphonic acid moiety.

Embodiment 7 the microfluidic device according to any one of embodiments 1 to 6, wherein at least one conditioned surface comprises a linking group covalently attached to a surface of the microfluidic device, and wherein the linking group is attached to a portion within the microfluidic device that is configured to support cell growth, viability, portability, or any combination thereof.

Embodiment 8 the microfluidic device according to embodiment 7, wherein the linker is a siloxy linker.

Embodiment 9. the microfluidic device according to embodiment 7 or 8, wherein at least one conditioned surface comprises an alkyl or fluoroalkyl moiety.

Embodiment 10. the microfluidic device according to embodiment 9, wherein the alkyl or fluoroalkyl moiety has a backbone length greater than 10 carbons.

Embodiment 11 the microfluidic device according to any one of embodiments 7 to 10, wherein the linking group is indirectly attached to the moiety configured to support cell growth, viability, portability, or any combination thereof via a linker.

Embodiment 12. the microfluidic device according to embodiment 11, wherein the linker comprises a triazolylene moiety.

Embodiment 13. the microfluidic device according to any one of embodiments 1 to 12, wherein at least one conditioned surface comprises a sugar moiety.

Embodiment 14 the microfluidic device according to any one of embodiments 1 to 13, wherein at least one conditioned surface comprises an alkylene ether moiety.

Embodiment 15 the microfluidic device according to any one of embodiments 1 to 14, wherein at least one conditioned surface comprises an amino acid moiety.

Embodiment 16 the microfluidic device according to any one of embodiments 7 to 15, wherein at least one conditioned surface comprises a zwitterion.

Embodiment 17 the microfluidic device according to any one of embodiments 1 to 16, wherein the conditioned surface comprises a cleavable moiety.

Embodiment 18. the microfluidic device according to any one of embodiments 1 to 17, wherein the microfluidic device further comprises a substrate having a Dielectrophoresis (DEP) configuration.

Embodiment 19 the microfluidic device according to embodiment 18, wherein the DEP configuration is optically actuated.

Embodiment 20 the microfluidic device according to any one of embodiments 1 to 19, wherein at least one growth chamber comprises at least one surface conditioned to support cell growth, viability, portability, or any combination thereof, of mammalian cells.

Embodiment 21. the microfluidic device according to any one of embodiments 1 to 20, wherein at least one growth chamber comprises at least one surface conditioned to support cell growth, viability, portability, or any combination thereof, of plant protoplasts.

Embodiment 22. the microfluidic device according to embodiment 21, wherein the plant protoplast is from an agricultural plant.

Embodiment 23. the microfluidic device according to embodiment 22, wherein said plant protoplasts are from a lettuce, tomato, maize, wheat or tobacco plant.

Embodiment 24. the microfluidic device according to any one of embodiments 1 to 23, wherein at least one growth chamber comprises at least one surface conditioned to support cell growth, viability, portability, or any combination thereof, of individual plant cells and corresponding clonal colonies of plant cells.

Embodiment 25. a method of culturing at least one plant protoplast cell in a microfluidic device having a flow region configured to accommodate a flow of a first fluid medium and at least one growth chamber, the method comprising the steps of: introducing at least one plant protoplast cell into the at least one growth chamber, wherein the at least one growth chamber is configured to have at least one surface conditioned to support growth, viability, portability, or any combination thereof of the cell; and incubating the at least one plant protoplast cell for a period of time at least long enough to expand the at least one plant protoplast cell to produce a plant protoplast cell colony.

Embodiment 26 the method of embodiment 25, wherein the microfluidic device is the microfluidic device of any one of embodiments 1 to 24.

Embodiment 27. the method of embodiment 25 or 26, further comprising: conditioning at least one surface of the at least one growth chamber.

Embodiment 28 the method of embodiment 27, wherein conditioning comprises treating at least one surface of at least one growth chamber with a conditioning agent comprising a polymer.

Embodiment 29. the method of any one of embodiments 25 to 28, wherein introducing the at least one plant protoplast cell into the at least one growth chamber comprises moving the at least one plant protoplast cell using a Dielectrophoretic (DEP) force of sufficient strength.

Embodiment 30 the method of embodiment 29, wherein the DEP force is optically actuated.

Embodiment 31. the method of any of embodiments 25 to 30, further comprising: perfusing a first fluid medium during the incubating step, wherein the first fluid medium is introduced via at least one inlet port of the microfluidic device and output via at least one outlet port of the microfluidic device, wherein, upon output, the first fluid medium optionally comprises a component from a second fluid medium.

Embodiment 32. the method of any of embodiments 25 to 31, further comprising: lysing the one or more cleavable moieties of the conditioned surface after the incubating step, thereby facilitating export of the one or more plant protoplast cells from the growth chamber or the isolation region thereof and into the flow region.

Embodiment 33. the method of any of embodiments 25 to 32, further comprising: one or more plant protoplast cells are transported out of the growth chamber or isolated region thereof and into the flow region.

Example 34. the method according to any one of examples 25 to 33, wherein the protoplasts are from lettuce, tomato, maize, wheat or tobacco plants.

Embodiment 35. the method of any one of embodiments 25 to 34, wherein introducing at least one plant protoplast cell into at least one growth chamber comprises introducing a single plant protoplast cell into the growth chamber, and wherein the plant protoplast cell colony generated by the incubating step is a clonal colony.

Embodiment 36. the method of any of embodiments 25 to 35, wherein the first fluid medium is a growth medium that supports protoplast growth.

Example 37. a method of identifying plant protoplasts that lack pathogen resistance, the method comprising: introducing a first fluid medium comprising one or more protoplasts into a microfluidic device comprising a housing having a flow region and at least one growth chamber; moving a first protoplast of the one or more protoplasts into a first growth chamber of the at least one growth chamber; contacting the first protoplast with a pathogen; and monitoring the viability of the first protoplast during a first time period after the first protoplast is contacted with the pathogen, wherein the viability of the protoplast at the end of the first time period indicates that the protoplast lacks resistance to the pathogen.

Embodiment 38. the method of embodiment 37, wherein the one or more protoplasts are from a large area crop.

Example 39. the method of example 38, wherein the large area crop is a wheat, corn, soybean or cotton plant.

Example 40. the method of example 37, wherein the one or more protoplasts are from a high value or ornamental crop.

Example 41. the method according to example 40, wherein the high value crop is a tomato, lettuce, pepper or squash plant.

Embodiment 42. the method of embodiment 37, wherein the one or more protoplasts are from a turf or a forage plant.

Example 43. the method according to example 42, wherein the turf or forage plant is a grass or alfalfa plant.

Example 44. the method of example 37, wherein the one or more protoplasts are from an experimental plant (e.g., an arabidopsis thaliana plant or a snapdragon plant).

Embodiment 45. the method according to any one of embodiments 37 to 44, wherein the pathogen is a plant pathogen or a molecule derived from a plant pathogen.

Example 46. the method according to example 45, wherein the plant pathogen is a virus, a bacterium or a fungal cell.

Example 47. the method of example 45 or 46, wherein the pathogen is a molecular agent (e.g., a viral capsid protein, flagellin, lipopolysaccharide, peptidoglycan, chitin protein) or a fragment thereof.

Embodiment 48. the method of any one of embodiments 37 to 47, wherein contacting the first protoplast with the pathogen comprises flowing a second fluid medium comprising the pathogen into a flow region of the microfluidic device.

Embodiment 49. the method of embodiment 48, wherein contacting the first protoplast with the pathogen further comprises moving the pathogen into the isolated region of the first growth chamber or allowing the pathogen to diffuse from the flow region to the isolated region of the first growth chamber.

Embodiment 50 the method of any one of embodiments 37-49, wherein the housing further comprises a base, a microfluidic circuit structure disposed on the base, and a cover.

Embodiment 51. the method of embodiment 50, wherein the lid and the base are part of a Dielectrophoresis (DEP) mechanism for selectively inducing a DEP force on a micro-object, and wherein moving the first protoplast into the first growth chamber comprises applying the DEP force on the first protoplast.

Embodiment 52. the method of any of embodiments 37-51, wherein the microfluidic device further comprises a first electrode, an electrode-activating substrate, and a second electrode, wherein the first electrode is part of a first wall of the housing, the electrode-activating substrate and the second electrode are part of a second wall of the housing, wherein the electrode-activating substrate comprises a photoconductive material, a semiconductor integrated circuit, or a phototransistor, and wherein moving the first protoplast into the first growth chamber comprises applying a DEP force on the first protoplast.

Embodiment 53. the method of embodiment 52, wherein the first wall is the lid, and wherein the second wall is the base.

Embodiment 54 the method of embodiment 52 or 53, wherein the electrode activation substrate comprises a phototransistor.

Embodiment 55. the method of embodiment 50 or 53, wherein the cover and/or the base are optically transparent.

Embodiment 56. the method of any one of embodiments 37 to 55, wherein the first growth chamber is an isolation pen comprising an isolation region and a communication region in fluid communication with the isolation region to the flow region, and wherein the isolation region is an unswept region of the microfluidic device.

Embodiment 57 the method of embodiment 56, wherein the housing further comprises a microfluidic channel comprising at least a portion of the flow region, wherein the communication region of the isolation fence comprisesA proximal opening into the microfluidic channel and a distal opening into the isolation region, the proximal opening having a width W_conIs about 50 microns to about 150 microns, and wherein the length L of the communication zone from the proximal opening to the distal opening_conIs the width W of the proximal opening of the communication region_conAt least 1.0 times.

Example 58. the method of example 57, wherein the length L of the communication zone from the proximal opening to the distal opening_conIs the width W of the proximal opening of the communication region_conAt least 1.5 times.

Example 59. the method of example 57, wherein the length L of the communication zone from the proximal opening to the distal opening_conIs the width W of the proximal opening of the communication region_conAt least 2.0 times.

Embodiment 60. the method of any of embodiments 57 to 59, wherein the width W of the proximal opening of the communication zone_conFrom about 50 microns to about 100 microns.

Embodiment 61. the method of any of embodiments 57 to 60, wherein the length L of the communication zone from the proximal opening to the distal opening_conFrom about 50 microns to about 500 microns.

Embodiment 62. the method of any of embodiments 57 to 61, wherein the height H of the microfluidic channel at the proximal opening of the communication region_chBetween 20 microns and 100 microns (e.g., between about 30 microns and 60 microns)).

Embodiment 63. the method of any one of embodiments 57 to 62, wherein the microfluidic channel has a width W at the proximal opening of the communication region_chBetween about 50 microns and about 500 microns (e.g., between about 100 microns and about 250 microns).

Embodiment 64. the method of any one of embodiments 56 to 63, wherein the volume of the isolation region of the isolation fence is about 5 x 10⁵To about 5X 10⁶Cubic microns.

Example 65 according to example 56The method of any one of claims to 64, wherein the volume of the isolation region of the isolation fence is about 1 x 10⁶To about 2X 10⁶Cubic microns.

Embodiment 66. the method of any of embodiments 56 to 65, wherein the proximal opening of the communication zone is parallel to the direction of bulk flow in the flow zone.

Embodiment 67. the method of any one of embodiments 37 to 66, wherein monitoring the viability of the first protoplast during the first time period comprises monitoring the first protoplast for cell division, and wherein cell division of the first protoplast indicates that the protoplast lacks resistance to the pathogen.

Embodiment 68. the method of any one of embodiments 37-67, wherein monitoring viability of the first protoplast during the first time period comprises maintaining the microfluidic chip at a temperature of about 20 ℃ to about 30 ℃ (e.g., about 24 ℃ to about 26 ℃) during the first time period and/or minimizing the amount of light to which the first protoplast is exposed during the first time period (e.g., by keeping the microfluidic chip in a dark environment or substantially blocking light outside the instrument from entering an isolation pen).

Embodiment 69. the method of any one of embodiments 37-68, wherein monitoring viability of the first protoplast during the first time period comprises periodically perfusing a protoplast growth medium through the flow region of the microfluidic device during the first time period.

Embodiment 70. the method of embodiment 69, wherein the protoplast growth medium is perfused no more than once a day (e.g., no more than once every two, three, four, five, six, seven, or more days) through the flow region.

Embodiment 71. the method of any one of embodiments 37 to 70, wherein monitoring viability of the first protoplasts during the first time period comprises staining the first protoplasts with a cell viability dye (e.g., fluorescein diacetate (i.e., FDA) or Hoechst).

Embodiment 72. the method of any one of embodiments 37 to 71, wherein monitoring viability of the first protoplasts during the first time period comprises staining the first protoplasts with a chlorophyll stain and/or a cell wall stain (e.g., an optical brightener).

Embodiment 73. the method of any one of embodiments 37 to 72, wherein the first period of time is at least 12 hours.

Embodiment 74. the method of embodiment 74, wherein the first period of time is at least 24, 48, 72, 96, 120 hours or more (e.g., 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days or more).

Embodiment 75. the method of any of embodiments 37 to 74, further comprising: determining that the first protoplast lacks resistance to the pathogen; and outputting the first protoplast from the first growth chamber and the microfluidic device.

Embodiment 76. the method of any one of embodiments 37 to 75, further comprising: determining that the first protoplast lacks resistance to the pathogen; and sequencing one or more disease resistance genes of the first protoplast.

Embodiment 77. the method according to any of embodiments 37-76, further comprising: determining that the first protoplast lacks resistance to the pathogen; and sequencing the transcriptome of the first protoplast.

Embodiment 78 the method of any one of embodiments 37 to 77, further comprising: determining that the first protoplast lacks resistance to the pathogen; and sequencing the genome of the first protoplast.

Embodiment 79. the method according to any of embodiments 76 to 78, further comprising: identifying sequences of one or more disease resistance genes, molecular changes or defects in the transcriptome and/or genome that are associated with the lack of pathogen resistance.

Embodiment 80. the method of any one of embodiments 37 to 79, further comprising: moving at least one protoplast into each of a plurality of growth chambers in the microfluidic device; and performing the remaining steps of the method on each protoplast that is moved into the plurality of growth chambers.

Example 81 a kit for screening plant protoplasts for disease resistance, the kit comprising: a microfluidic chip, wherein the microfluidic chip comprises a housing having a flow region and at least one growth chamber; and a reagent for detecting the viability of the plant protoplast.

Example 82. a kit according to example 81, comprising a surface conditioning agent.

Embodiment 83. the kit of embodiment 81 or 82, further comprising a conditioning modifier, and wherein at least one surface of the growth chamber comprises a surface modifying ligand.

Embodiment 84. the kit of embodiment 81 or 82, wherein at least one surface of the growth chamber comprises a covalently attached coating material.

Embodiment 85. the kit of any one of embodiments 81 to 84, wherein the reagent for detecting viability of plant protoplasts is a fluorescent stain (e.g., Fluorescein Diacetate (FDA), Hoechst, fluorescent whitening agent, chlorophyll stain, etc.).